17Q-linked breast and ovarian cancer susceptibility gene

ABSTRACT

The present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect a human breast and ovarian cancer predisposing gene (BRCA1), some mutant alleles of which cause susceptibility to cancer, in particular breast and ovarian cancer. More specifically, the invention relates to germline mutations in the BRCA1 gene and their use in the diagnosis of predisposition to breast and ovarian cancer. The present invention further relates to somatic mutations in the BRCA1 gene in human breast and ovarian cancer and their use in the diagnosis and prognosis of human breast and ovarian cancer. Additionally, the invention relates to somatic mutations in the BRCA1 gene in other human cancers and their use in the diagnosis and prognosis of human cancers. The invention also relates to the therapy of human cancers which have a mutation in the BRCA1 gene, including gene therapy, protein replacement therapy and protein mimetics. The invention further relates to the screening of drugs for cancer therapy. Finally, the invention relates to the screening of the BRCA1 gene for mutations, which are useful for diagnosing the predisposition to breast and ovarian cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/409,305 filed on 24 Mar. 1995, abandoned, which is acontinuation-in-part of application Ser. No. 08/348,824 filed on 29 Nov.1994, abandoned, which is a continuation-in-part of application Ser. No.08/308,104 filed on 16 Sep. 1994, which is a continuation-in-part ofapplication Ser. No. 08/300,266, filed on 2 Sep. 1994, abandoned, whichis a continuation-in-part of application Ser. No. 08/289,221, filed on12 Aug. 1994, abandoned, all incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the field of human genetics.Specifically, the present invention relates to methods and materialsused to isolate and detect a human breast and ovarian cancerpredisposing gene (BRCA1), some mutant alleles of which causesusceptibility to cancer, in particular, breast and ovarian cancer. Morespecifically, the invention relates to germline mutations in the BRCA1gene and their use in the diagnosis of predisposition to breast andovarian cancer. The present invention further relates to somaticmutations in the BRCA1 gene in human breast and ovarian cancer and theiruse in the diagnosis and prognosis of human breast and ovarian cancer.Additionally, the invention relates to somatic mutations in the BRCA1gene in other human cancers and their use in the diagnosis and prognosisof human cancers. The invention also relates to the therapy of humancancers which have a mutation in the BRCA1 gene, including gene therapy,protein replacement therapy and protein mimetics. The invention furtherrelates to the screening of drugs for cancer therapy. Finally, theinvention relates to the screening of the BRCA1 gene for mutations,which are useful for diagnosing the predisposition to breast and ovariancancer.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated herein byreference, and for convenience, are referenced by author and date in thefollowing text and respectively grouped in the appended List ofReferences.

BACKGROUND OF THE INVENTION

The genetics of cancer is complicated, involving multiple dominant,positive regulators of the transformed state (oncogenes) as well asmultiple recessive, negative regulators (tumor suppressor genes). Overone hundred oncogenes have been characterized. Fewer than a dozen tumorsuppressor genes have been identified, but the number is expected toincrease beyond fifty (Knudson, 1993).

The involvement of so many genes underscores the complexity of thegrowth control mechanisms that operate in cells to maintain theintegrity of normal tissue. This complexity is manifest in another way.So far, no single gene has been shown to participate in the developmentof all, or even the majority of human cancers. The most common oncogenicmutations are in the H-ras gene, found in 10-15% of all solid tumors(Anderson et al., 1992). The most frequently mutated tumor suppressorgenes are the TP53 gene, homozygously deleted in roughly 50% of alltumors, and CDKN2, which was homozygously deleted in 46% of tumor celllines examined (Kamb et al., 1994). Without a target that is common toall transformed cells, the dream of a "magic bullet" that can destroy orrevert cancer cells while leaving normal tissue unharmed is improbable.The hope for a new generation of specifically targeted antitumor drugsmay rest on the ability to identify tumor suppressor genes or oncogenesthat play general roles in control of cell division.

The tumor suppressor genes which have been cloned and characterizedinfluence susceptibility to: 1) Retinoblastoma (RB1); 2) Wilms' tumor(WT1); 3) Li-Fraumeni (TP53); 4) Familial adenomatus polyposis (APC); 5)Neurofibromatosis type 1 (NF1); 6) Neurofibromatosis type 2 (NF2); 7 vonHippel-Lindau syndrome (VHL); 8) Multiple endocrine neoplasia type 2A(MEN2A); and 9) Melanoma (CDKN2).

Tumor suppressor loci that have been mapped genetically but not yetisolated include genes for: Multiple endocrine neoplasia type 1 (MEN1);Lynch cancer family syndrome 2 (LCFS2); Neuroblastoma (NB); Basal cellnevus syndrome (BCNS); Beckwith-Wiedemann syndrome (BWS); Renal cellcarcinoma (RCC); Tuberous sclerosis 1 (TSC1); and Tuberous sclerosis 2(TSC2). The tumor suppressor genes that have been characterized to dateencode products with similarities to a variety of protein types,including DNA binding proteins (WT1), ancillary transcription regulators(RB1), GTPase activating proteins or GAPs (NF1), cytoskeletal components(NF2), membrane bound receptor kinases (MEN2A), cell cycle regulators(CDKN2) and others with no obvious similarity to known proteins (APC andVHL).

In many cases, the tumor suppressor gene originally identified throughgenetic studies has been shown to be lost or mutated in some sporadictumors. This result suggests that regions of chromosomal aberration maysignify the position of important tumor suppressor genes involved bothin genetic predisposition to cancer and in sporadic cancer.

One of the hallmarks of several tumor suppressor genes characterized todate is that they are deleted at high frequency in certain tumor types.The deletions often involve loss of a single allele, a so-called loss ofheterozygosity (LOH), but may also involve homozygous deletion of bothalleles. For LOH, the remaining allele is presumed to be nonfunctional,either because of a preexisting inherited mutation, or because of asecondary sporadic mutation.

Breast cancer is one of the most significant diseases that affectswomen. At the current rate, American women have a 1 in 8 risk ofdeveloping breast cancer by age 95 (American Cancer Society, 1992).Treatment of breast cancer at later stages is often futile anddisfiguring, making early detection a high priority in medicalmanagement of the disease. Ovarian cancer, although less frequent thanbreast cancer is often rapidly fatal and is the fourth most common causeof cancer mortality in American women. Genetic factors contribute to anill-defined proportion of breast cancer incidence, estimated to be about5% of all cases but approximately 25% of cases diagnosed before age 40(Claus et al., 1991). Breast cancer has been subdivided into two types,early-age onset and late-age onset, based on an inflection in theage-specific incidence curve around age 50. Mutation of one gene, BRCA1,is thought to account for approximately 45% of familial breast cancer,but at least 80% of families with both breast and ovarian cancer (Eastonet al., 1993).

Intense efforts to isolate the BRCA1 gene have proceeded since it wasfirst mapped in 1990 (Hall et al., 1990; Narod et al., 1991). A secondlocus, BRCA2, has recently been mapped to chromosome 13q (Wooster etal., 1994) and appears to account for a proportion of early-onset breastcancer roughly equal to BRCA1, but confers a lower risk of ovariancancer. The remaining susceptibility to early-onset breast cancer isdivided between as yet unmapped genes for familial cancer, and rarergermline mutations in genes such as TP53 (Malkin et al., 1990). It hasalso been suggested that heterozygote carriers for defective forms ofthe Ataxia-Telangectasia gene are at higher risk for breast cancer(Swift et al., 1976; Swift et al., 1991). Late-age onset breast canceris also often familial although the risks in relatives are not as highas those for early-onset breast cancer (Cannon-Albright et al., 1994;Mettlin et al., 1990). However, the percentage of such cases due togenetic susceptibility is unknown.

Breast cancer has long been recognized to be, in part, a familialdisease (Anderson, 1972). Numerous investigators have examined theevidence for genetic inheritance and concluded that the data are mostconsistent with dominant inheritance for a major susceptibility locus orloci (Bishop and Gardner, 1980; Go et al., 1983; Willams and Anderson,1984; Bishop et al., 1988; Newman et al., 1988; Claus et al., 1991).Recent results demonstrate that at least three loci exist which conveysusceptibility to breast cancer as well as other cancers. These loci arethe TP53 locus on chromosome 17p (Malkin et al., 1990), a 17q-linkedsusceptibility locus known as BRCA1 (Hall et al., 1990), and one or moreloci responsible for the unmapped residual. Hall et al. (1990) indicatedthat the inherited breast cancer susceptibility in kindreds with earlyage onset is linked to chromosome 17q21; although subsequent studies bythis group using a more appropriate genetic model partially refuted thelimitation to early onset breast cancer (Margaritte et al., 1992).

Most strategies for cloning the 17q-linked breast cancer predisposinggene (BRCA1) require precise genetic localization studies. The simplestmodel for the functional role of BRCA1 holds that alleles of BRCA1 thatpredispose to cancer are recessive to wild type alleles; that is, cellsthat contain at least one wild type BRCA1 allele are not cancerous.However, cells that contain one wild type BRCA1 allele and onepredisposing allele may occasionally suffer loss of the wild type alleleeither by random mutation or by chromosome loss during cell division(nondisjunction). All the progeny of such a mutant cell lack the wildtype function of BRCA1 and may develop into tumors. According to thismodel, predisposing alleles of BRCA1 are recessive, yet susceptibilityto cancer is inherited in a dominant fashion: women who possess onepredisposing allele (and one wild type allele) risk developing cancer,because their mammary epithelial cells may spontaneously lose the wildtype BRCA1 allele. This model applies to a group of cancersusceptibility loci known as tumor suppressors or antioncogenes, a classof genes that includes the retinoblastoma gene and neurofibromatosisgene. By inference this model may also explain the BRCA1 function, ashas recently been suggested (Smith et al., 1992).

A second possibility is that BRCA1 predisposing alleles are trulydominant; that is, a wild type allele of BRCA1 cannot overcome the tumorforming role of the predisposing allele. Thus, a cell that carries bothwild type and mutant alleles would not necessarily lose the wild typecopy of BRCA1 before giving rise to malignant cells. Instead, mammarycells in predisposed individuals would undergo some other stochasticchange(s) leading to cancer.

If BRCA1 predisposing alleles are recessive, the BRCA1 gene is expectedto be expressed in normal mammary tissue but not functionally expressedin mammary tumors. In contrast, if BRCA1 predisposing alleles aredominant, the wild type BRCA1 gene may or may not be expressed in normalmammary tissue. However, the predisposing allele will likely beexpressed in breast tumor cells.

The 17q linkage of BRCA1 was independently confirmed in three of fivekindreds with both breast cancer and ovarian cancer (Narod et al.,1991). These studies claimed to localize the gene within a very largeregion, 15 centiMorgans (cM), or approximately 15 million base pairs, toeither side of the linked marker pCMM86 (D17S74). However, attempts todefine the region further by genetic studies, using markers surroundingpCMMS6, proved unsuccessful. Subsequent studies indicated that the genewas considerably more proximal (Easton et al., 1993) and that theoriginal analysis was flawed (Margaritte et al., 1992). Hall et al.,(1992) recently localized the BRCA1 gene to an approximately 8 cMinterval (approximately 8 million base pairs) bounded by Mfd15 (D17S250)on the proximal side and the human GIP gene on the distal side. Aslightly narrower interval for the BRCA1 locus, based on publiclyavailable data, was agreed upon at the Chromosome 17 workshop in Marchof 1992 (Fain, 1992). The size of these regions and the uncertaintyassociated with them has made it exceedingly difficult to design andimplement physical mapping and/or cloning strategies for isolating theBRCA1 gene.

Identification of a breast cancer susceptibility locus would permit theearly detection of susceptible individuals and greatly increase ourability to understand the initial steps which lead to cancer. Assusceptibility loci are often altered during tumor progression, cloningthese genes could also be important in the development of betterdiagnostic and prognostic products, as well as better cancer therapies.

SUMMARY OF THE INVENTION

The present invention relates generally to the field of human genetics.Specifically, the present invention relates to methods and materialsused to isolate and detect a human breast cancer predisposing gene(BRCA1), some alleles of which cause susceptibility to cancer, inparticular breast and ovarian cancer. More specifically, the presentinvention relates to germline mutations in the BRCA1 gene and their usein the diagnosis of predisposition to breast and ovarian cancer. Theinvention further relates to somatic mutations in the BRCA1 gene inhuman breast cancer and their use in the diagnosis and prognosis ofhuman breast and ovarian cancer. Additionally, the invention relates tosomatic mutations in the BRCA1 gene in other human cancers and their usein the diagnosis and prognosis of human cancers. The invention alsorelates to the therapy of human cancers which have a mutation in theBRCA1 gene, including gene therapy, protein replacement therapy andprotein mimetics. The invention further relates to the screening ofdrugs for cancer therapy. Finally, the invention relates to thescreening of the BRCA1 gene for mutations, which are useful fordiagnosing the predisposition to breast and ovarian cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the order of loci neighboring BRCA1 asdetermined by the chromosome 17 workshop. FIG. 1 is reproduced fromFain, 1992.

FIG. 2 is a schematic map of YACs which define part of Mfd15-Mfd188region.

FIG. 3 is a schematic map of STSs, P1s and BACs in the BRCA1 region.

FIG. 4 is a schematic map of human chromosome 17. The pertinent regioncontaining BRCA1 is expanded to indicate the relative positions of twopreviously identified genes, CA125 and RNU2, BRCA1 spans the markerD17S855.

FIG. 5 shows alignment of the BRCA1 zinc-finger domain with 3 otherzinc-finger domains that scored highest in a Smith-Waterman alignment.RPT1 encodes a protein that appears to be a negative regulator of theIL-2 receptor in mouse. RIN1 encodes a DNA-binding protein that includesa RING-finger motif related to the zinc-finger. RFP1 encodes a putativetranscription factor that is the N-terminal domain of the RET oncogeneproduct. The bottom line contains the C3HC4 consensus zinc-fingersequence showing the positions of cysteines and one histidine that formthe zinc ion binding pocket.

FIG. 6 is a diagram of BRCA1 mRNA showing the locations of introns andthe variants of BRCA1 mRNA produced by alternative splicing. Intronlocations are shown by dark triangles and the exons are numbered belowthe line representing the cDNA. The top cDNA is the composite used togenerate the peptide sequence of BRCA1. Alternative forms identified ascDNA clones or hybrid selection clones are shown below.

FIG. 7 shows the tissue expression pattern of BRCA1. The blot wasobtained from Clontech and contains RNA from the indicated tissues.Hybridization conditions were as recommended by the manufacturer using aprobe consisting of nucleotide positions 3631 to 3930 of BRCA1. Notethat both breast and ovary are heterogeneous tissues and the percentageof relevant epithelial cells can be variable. Molecular weight standardsare in kilobases.

FIG. 8 is a diagram of the 5' untranslated region plus the beginning ofthe translated region of BRCA1 showing the locations of introns and thevariants of BRCA1 mRNA produced by alternative splicing. Intronlocations are shown by broken dashed lines. Six alternate splice formsare shown.

FIG. 9A shows a nonsense mutation in Kindred 2082. P indicates theperson originally screened, b and c are haplotype carriers, a, d, e, f,and g do not carry the BRCA1 haplotype. The C to T mutation results in astop codon and creates a site for the restriction enzyme AvrII. PCRamplification products are cut with this enzyme. The carriers areheterozygous for the site and therefore show three bands. Non-carriersremain uncut.

FIG. 9B shows a mutation and cosegregation analysis in BRCA1 kindreds.Carrier individuals are represented as filled circles and squares in thepedigree diagrams. Frameshift mutation in Kindred 1910. The first threelanes are control, noncarrier samples. Lanes labeled 1-3 containsequences from carrier individuals. Lane 4 contains DNA from a kindredmember who does not carry the BRCA1 mutation. The diamond is used toprevent identification of the kindred. The frameshift resulting from theadditional C is apparent in lanes labeled 1, 2, and 3.

FIG. 9C shows a mutation and cosegregation analysis in BRCA1 kindreds.Carrier individuals are represented as filled circles and squares in thepedigree diagrams. Inferred regulatory mutation in Kindred 2035. ASOanalysis of carriers and noncarriers of 2 different polymorphisms (PM1and PM7) which were examined for heterozygosity in the germline andcompared to the heterozygosity of lymphocyte mRNA. The top 2 rows ofeach panel contain PCR products amplified from genomic DNA and thebottom 2 rows contain PCR products amplified from cDNA. "A" and "G" arethe two alleles detected by the ASO. The dark spots indicate that aparticular allele is present in the sample. The first three lanes of PM7represent the three genotypes in the general population.

FIG. 10A-10H show genomic sequence of BRCA1. The lower case lettersdenote intron sequence while the upper case letters denote exonsequence. Indefinite intervals within introns are designated withvvvvvvvvvvvvv. Known polymorphic sites are shown as underlined andboldface type.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to the field of human genetics.Specifically, the present invention relates to methods and materialsused to isolate and detect a human breast cancer predisposing gene(BRCA1), some alleles of which cause susceptibility to cancer, inparticular breast and ovarian cancer. More specifically, the presentinvention relates to germline mutations in the BRCA1 gene and their usein the diagnosis of predisposition to breast and ovarian cancer. Theinvention further relates to somatic mutations in the BRCA 1 gene inhuman breast cancer and their use in the diagnosis and prognosis ofhuman breast and ovarian cancer. Additionally, the invention relates tosomatic mutations in the BRCA1 gene in other human cancers and their usein the diagnosis and prognosis of human cancers. The invention alsorelates to the therapy of human cancers which have a mutation in theBRCA1 gene, including gene therapy, protein replacement therapy andprotein mimetics. The invention further relates to the screening ofdrugs for cancer therapy. Finally, the invention relates to thescreening of the BRCA1 gene for mutations, which are useful fordiagnosing the predisposition to breast and ovarian cancer.

The present invention provides an isolated polynucleotide comprisingall, or a portion of the BRCA1 locus or of a mutated BRCA1 locus,preferably at least eight bases and not more than about 100 kb inlength. Such polynucleotides may be antisense polynucleotides. Thepresent invention also provides a recombinant construct comprising suchan isolated polynucleotide, for example, a recombinant constructsuitable for expression in a transformed host cell.

Also provided by the present invention are methods of detecting apolynucleotide comprising a portion of the BRCA1 locus or its expressionproduct in an analyte. Such methods may further comprise the step ofamplifying the portion of the BRCA1 locus, and may further include astep of providing a set of polynucleotides which are primers foramplification of said portion of the BRCA1 locus. The method is usefulfor either diagnosis of the predisposition to cancer or the diagnosis orprognosis of cancer.

The present invention also provides isolated antibodies, preferablymonoclonal antibodies, which specifically bind to an isolatedpolypeptide comprised of at least five amino acid residues encoded bythe BRCA1 locus.

The present invention also provides kits for detecting in an analyte apolynucleotide comprising a portion of the BRCA1 locus, the kitscomprising a polynucleotide complementary to the portion of the BRCA1locus packaged in a suitable container, and instructions for its use.

The present invention further provides methods of preparing apolynucleotide comprising polymerizing nucleotides to yield a sequencecomprised of at least eight consecutive nucleotides of the BRCA1 locus;and methods of preparing a polypeptide comprising polymerizing aminoacids to yield a sequence comprising at least five amino acids encodedwithin the BRCA1 locus.

The present invention further provides methods of screening the BRCA1gene to identify mutations. Such methods may further comprise the stepof amplifying a portion of the BRCA1 locus, and may further include astep of providing a set of polynucleotides which are primers foramplification of said portion of the BRCA1 locus. The method is usefulfor identifying mutations for use in either diagnosis of thepredisposition to cancer or the diagnosis or prognosis of cancer.

The present invention further provides methods of screening suspectedBRCA1 mutant alleles to identify mutations in the BRCA1 gene.

In addition, the present invention provides methods of screening drugsfor cancer therapy to identify suitable drugs for restoring BRCA1 geneproduct function.

Finally, the present invention provides the means necessary forproduction of gene-based therapies directed at cancer cells. Thesetherapeutic agents may take the form of polynucleotides comprising allor a portion of the BRCA1 locus placed in appropriate vectors ordelivered to target cells in more direct ways such that the function ofthe BRCA1 protein is reconstituted. Therapeutic agents may also take theform of polypeptides based on either a portion of, or the entire proteinsequence of BRCA1. These may functionally replace the activity of BRCA1in vivo.

It is a discovery of the present invention that the BRCA1 locus whichpredisposes individuals to breast cancer and ovarian cancer, is a geneencoding a BRCA1 protein, which has been found to have no significanthomology with known protein or DNA sequences. This gene is termed BRCA1herein. It is a discovery of the present invention that mutations in theBRCA1 locus in the germline are indicative of a predisposition to breastcancer and ovarian cancer. Finally, it is a discovery of the presentinvention that somatic mutations in the BRCA1 locus are also associatedwith breast cancer, ovarian cancer and other cancers, which representsan indicator of these cancers or of the prognosis of these cancers. Themutational events of the BRCA1 locus can involve deletions, insertionsand point mutations within the coding sequence and the non-codingsequence.

Starting from a region on the long arm of human chromosome 17 of thehuman genome, 17q, which has a size estimated at about 8 million basepairs, a region which contains a genetic locus, BRCA1, which causessusceptibility to cancer, including breast and ovarian cancer, has beenidentified.

The region containing the BRCA1 locus was identified using a variety ofgenetic techniques. Genetic mapping techniques initially defined theBRCA1 region in terms of recombination with genetic markers. Based uponstudies of large extended families ("kindreds") with multiple cases ofbreast cancer (and ovarian cancer cases in some kindreds), a chromosomalregion has been pinpointed that contains the BRCA1 gene as well as otherputative susceptibility alleles in the BRCA1 locus. Two meioticbreakpoints have been discovered on the distal side of the BRCA1 locuswhich are expressed as recombinants between genetic markers and thedisease, and one recombinant on the proximal side of the BRCA1 locus.Thus, a region which contains the BRCA1 locus is physically bounded bythese markers.

The use of the genetic markers provided by this invention allowed theidentification of clones which cover the region from a human yeastartificial chromosome (YAC) or a human bacterial artificial chromosome(BAC) library. It also allowed for the identification and preparation ofmore easily manipulated cosmid, P1 and BAC clones from this region andthe construction of a contig from a subset of the clones. These cosmids,P1s, YACs and BACs provide the basis for cloning the BRCA1 locus andprovide the basis for developing reagents effective, for example, in thediagnosis and treatment of breast and/or ovarian cancer. The BRCA1 geneand other potential susceptibility genes have been isolated from thisregion. The isolation was done using software trapping (a computationalmethod for identifying sequences likely to contain coding exons, fromcontiguous or discontinuous genomic DNA sequences), hybrid selectiontechniques and direct screening, with whole or partial cDNA inserts fromcosmids, P1s and BACs, in the region to screen cDNA libraries. Thesemethods were used to obtain sequences of loci expressed in breast andother tissue. These candidate loci were analyzed to identify sequenceswhich confer cancer susceptibility. We have discovered that there aremutations in the coding sequence of the BRCA1 locus in kindreds whichare responsible for the 17q-linked cancer susceptibility known as BRCA1.This gene was not known to be in this region. The present invention notonly facilitates the early detection of certain cancers, so vital topatient survival, but also permits the detection of susceptibleindividuals before they develop cancer.

Population Resources

Large, well-documented Utah kindreds are especially important inproviding good resources for human genetic studies. Each large kindredindependently provides the power to detect whether a BRCA1susceptibility allele is segregating in that family. Recombinantsinformative for localization and isolation of the BRCA1 locus could beobtained only from kindreds large enough to confirm the presence of asusceptibility allele. Large sibships are especially important forstudying breast cancer, since penetrance of the BRCA1 susceptibilityallele is reduced both by age and sex, making informative sibshipsdifficult to find. Furthermore, large sibships are essential forconstructing haplotypes of deceased individuals by inference from thehaplotypes of their close relatives.

While other populations may also provide beneficial information, suchstudies generally require much greater effort, and the families areusually much smaller and thus less informative. Utah's age-adjustedbreast cancer incidence is 20% lower than the average U.S. rate. Thelower incidence in Utah is probably due largely to an early age at firstpregnancy, increasing the probability that cases found in Utah kindredscarry a genetic predisposition.

Genetic Mapping

Given a set of informative families, genetic markers are essential forlinking a disease to a region of a chromosome. Such markers includerestriction fragment length polymorphisms (RFLPs) (Botstein et al.,1980), markers with a variable number of tandem repeats (VNTRs)(Jeffreys et al., 1985; Nakamura et al., 1987), and an abundant class ofDNA polymorphisms based on short tandem repeats (STRs), especiallyrepeats of CpA (Weber and May, 1989; Litt et al., 1989). To generate agenetic map, one selects potential genetic markers and tests them usingDNA extracted from members of the kindreds being studied.

Genetic markers useful in searching for a genetic locus associated witha disease can be selected on an ad hoc basis, by densely covering aspecific chromosome, or by detailed analysis of a specific region of achromosome. A preferred method for selecting genetic markers linked witha disease involves evaluating the degree of informativeness of kindredsto determine the ideal distance between genetic markers of a givendegree of polymorphism, then selecting markers from known genetic mapswhich are ideally spaced for maximal efficiency. Informativeness ofkindreds is measured by the probability that the markers will beheterozygous in unrelated individuals. It is also most efficient to useSTR markers which are detected by amplification of the target nucleicacid sequence using PCR; such markers are highly informative, easy toassay (Weber and May, 1989), and can be assayed simultaneously usingmultiplexing strategies (Skolnick and Wallace, 1988), greatly reducingthe number of experiments required.

Once linkage has been established, one needs to find markers that flankthe disease locus, i.e., one or more markers proximal to the diseaselocus, and one or more markers distal to the disease locus. Wherepossible, candidate markers can be selected from a known genetic map.Where none is known, new markers can be identified by the STR technique,as shown in the Examples.

Genetic mapping is usually an iterative process. In the presentinvention, it began by defining flanking genetic markers around theBRCA1 locus, then replacing these flanking markers with other markersthat were successively closer to the BRCA1 locus. As an initial step,recombination events, defined by large extended kindreds, helpedspecifically to localize the BRCA1 locus as either distal or proximal toa specific genetic marker (Goldgar et al., 1994).

The region surrounding BRCA1, until the disclosure of the presentinvention, was not well mapped and there were few markers. Therefore,short repetitive sequences on cosmids subcloned from YACs, which hadbeen physically mapped, were analyzed in order to develop new geneticmarkers. Using this approach, one marker of the present invention, 42D6,was discovered which replaced pCMM86 as the distal flanking marker forthe BRCA1 region. Since 42D6 is approximately 14 cM from pCMM86, theBRCA1 region was thus reduced by approximately 14 centiMorgans (Eastonet al., 1993). The present invention thus began by finding a much moreclosely linked distal flanking marker of the BRCA1 region. BRCA1 wasthen discovered to be distal to the genetic marker Mfd15. Therefore,BRCA1 was shown to be in a region of 6 to 10 million bases bounded byMfd15 and 42D6. Marker Mfd191was subsequently discovered to be distal toMfd15 and proximal to BRCA1. Thus, Mfd15 was replaced with Mfd191 as theclosest proximal genetic marker. Similarly, it was discovered thatgenetic marker Mfd188 could replace genetic marker 42D6, narrowing theregion containing the BRCA1 locus to approximately 1.5 million bases.Then the marker Mfd191 was replaced with tdj1474 as the proximal markerand Mfd188 was replaced with U5R as the distal marker, further narrowingthe BRCA1 region to a small enough region to allow isolation andcharacterization of the BRCA1 locus (see FIG. 3), using techniques knownin the art and described herein.

Physical Mapping

Three distinct methods were employed to physically map the region. Thefirst was the use of yeast artificial chromosomes (YACs) to clone theregion which is flanked by tdj1474 and U5R. The second was the creationof a set of P1, BAC and cosmid clones which cover the region containingthe BRCA1 locus.

Yeast Artificial Chromosomes (YACs). Once a sufficiently small regioncontaining the BRCA1 locus was identified, physical isolation of the DNAin the region proceeded by identifying a set of overlapping YACs whichcovers the region. Useful YACs can be isolated from known libraries,such as the St. Louis and CEPH YAC libraries, which are widelydistributed and contain approximately 50,000 YACs each. The YACsisolated were from these publicly accessible libraries and can beobtained from a number of sources including the Michigan Genome Center.Clearly, others who had access to these YACs, without the disclosure ofthe present invention, would not have known the value of the specificYACs we selected since they would not have known which YACs were within,and which YACs outside of, the smallest region containing the BRCA1locus.

Cosmid, P1 and BAC Clones. In the present invention, it is advantageousto proceed by obtaining cosmid, P1, and BAC clones to cover this region.The smaller size of these inserts, compared to YAC inserts, makes themmore useful as specific hybridization probes. Furthermore, having thecloned DNA in bacterial cells, rather than in yeast cells, greatlyincreases the ease with which the DNA of interest can be manipulated,and improves the signal-to-noise ratio of hybridization assays. Forcosmid subclones of YACs, the DNA is partially digested with therestriction enzyme Sau3A and cloned into the BamHI site of the pWE15cosmid vector (Stratagene, cat. #1251201). The cosmids containing humansequences are screened by hybridization with human repetitive DNA (e.g.,Gibco/BRL, Human C_(o) t-1 DNA, cat. 5279SA), and then fingerprinted bya variety of techniques, as detailed in the Examples.

P1 and BAC clones are obtained by screening libraries constructed fromthe total human genome with specific sequence tagged sites (STSs)derived from the YACs, cosmids or P1s and BACs, isolated as describedherein.

These P1, BAC and cosmid clones can be compared by interspersedrepetitive sequence (IRS) PCR and/or restriction enzyme digests followedby gel electrophoresis and comparison of the resulting DNA fragments("fingerprints") (Maniatis et al., 1982). The clones can also becharacterized by the presence of STSs. The fingerprints are used todefine an overlapping contiguous set of clones which covers the regionbut is not excessively redundant, referred to herein as a "minimumtiling path". Such a minimum tiling path forms the basis for subsequentexperiments to identify cDNAs which may originate from the BRCA1 locus.

Coverage of the Gap with P1 and BAC Clones. To cover any gaps in theBRCA1 contig between the identified cosmids with genomic clones, clonesin P1 and BAC vectors which contain inserts of genomic DNA roughly twiceas large as cosmids for P1s and still greater for BACs (Sternberg, 1990;Sternberg et al., 1990; Pierce et al., 1992; Shizuya et al., 1992) wereused. P1 clones were isolated by Genome Sciences using PCR primersprovided by us for screening. BACs were provided by hybridizationtechniques in Dr. Mel Simon's laboratory. The strategy of using P1clones also permitted the covering of the genomic region with anindependent set of clones not derived from YACs. This guards against thepossibility of other deletions in YACs that have not been detected.These new sequences derived from the P1 clones provide the material forfurther screening for candidate genes, as described below.

Gene Isolation.

There are many techniques for testing genomic clones for the presence ofsequences likely to be candidates for the coding sequence of a locus oneis attempting to isolate, including but not limited to:

a. zoo blots

b. identifying HTF islands

c. exon trapping

d. hybridizing cDNA to cosmids or YACs.

e. screening cDNA libraries.

(a) Zoo blots. The first technique is to hybridize cosmids to Southernblots to identify DNA sequences which are evolutionarily conserved, andwhich therefore give positive hybridization signals with DNA fromspecies of varying degrees of relationship to humans (such as monkey,cow, chicken, pig, mouse and rat). Southern blots containing such DNAfrom a variety of species are commercially available (Clonetech, Cat.7753-1).

(b) Identifing HTF islands. The second technique involves findingregions rich in the nucleotides C and G, which often occur near orwithin coding sequences. Such sequences are called HTF (HpaI tinyfragment) or CpG islands, as restriction enzymes specific for siteswhich contain CpG dimers cut frequently in these regions (Lindsay etal., 1987).

(c) Exon trapping. The third technique is exon trapping, a method thatidentifies sequences in genomic DNA which contain splice junctions andtherefore are likely to comprise coding sequences of genes. Exonamplification (Buckler et al., 1991) is used to select and amplify exonsfrom DNA clones described above. Exon amplification is based on theselection of RNA sequences which are flanked by functional 5' and/or 3'splice sites. The products of the exon amplification are used to screenthe breast cDNA libraries to identify a manageable number of candidategenes for further study. Exon trapping can also be performed on smallsegments of sequenced DNA using computer programs or by softwaretrapping.

(d) Hybridizing cDNA to Cosmids, P1s, BACs or YACs. The fourth techniqueis a modification of the selective enrichment technique which utilizeshybridization of cDNA to cosmids, P1s, BACs or YACs and permitstranscribed sequences to be identified in, and recovered from clonedgenomic DNA (Kandpal et al, 1990). The selective enrichment technique,as modified for the present purpose, involves binding DNA from theregion of BRCA1 present in a YAC to a column matrix and selecting cDNAsfrom the relevant libraries which hybridize with the bound DNA, followedby amplification and purification of the bound DNA, resulting in a greatenrichment for cDNAs in the region represented by the cloned genomicDNA.

(e) Identification of cDNAs. The fifth technique is to identify cDNAsthat correspond to the BRCA1 locus. Hybridization probes containingputative coding sequences, selected using any of the above techniques,are used to screen various libraries, including breast tissue cDNAlibraries, ovarian cDNA libraries, and any other necessary libraries.

Another variation on the theme of direct selection of cDNA was also usedto find candidate genes for BRCA1 (Lovett et al., 1991; Futreal, 1993).This method uses cosmid, P1 or BAC DNA as the probe. The probe DNA isdigested with a blunt cutting restriction enzyme such as HaeIII. Doublestranded adapters are then ligated onto the DNA and serve as bindingsites for primers in subsequent PCR amplification reactions usingbiotinylated primers. Target cDNA is generated from mRNA derived fromtissue samples, e.g., breast tissue, by synthesis of either randomprimed or oligo(dT) primed first strand followed by second strandsynthesis. The cDNA ends are rendered blunt and ligated ontodouble-stranded adapters. These adapters serve as amplification sitesfor PCR. The target and probe sequences are denatured and mixed withhuman C_(o) t-1 DNA to block repetitive sequences.

Solution hybridization is carried out to high C_(o) t-1/2 values toensure hybridization of rare target cDNA molecules. The annealedmaterial is then captured on avidin beads, washed at high stringency andthe retained cDNAs are eluted and amplified by PCR. The selected cDNA issubjected to further rounds of enrichment before cloning into a plasmidvector for analysis.

Testing the cDNA for Candidacy

Proof that the cDNA is the BRCA1 locus is obtained by finding sequencesin DNA extracted from affected kindred members which create abnormalBRCA1 gene products or abnormal levels of BRCA1 gene product. Such BRCA1susceptibility alleles will co-segregate with the disease in largekindreds. They will also be present at a much higher frequency innon-kindred individuals with breast and ovarian cancer then inindividuals in the general population. Finally, since tumors oftenmutate somatically at loci which are in other instances mutated in thegermline, we expect to see normal germline BRCA1 alleles mutated intosequences which are identical or similar to BRCA1 susceptibility allelesin DNA extracted from tumor tissue. Whether one is comparing BRCA1sequences from tumor tissue to BRCA1 alleles from the germline of thesame individuals, or one is comparing germline BRCA1 alleles from cancercases to those from unaffected individuals, the key is to find mutationswhich are serious enough to cause obvious disruption to the normalfunction of the gene product. These mutations can take a number offorms. The most severe forms would be frame shift mutations or largedeletions which would cause the gene to code for an abnormal protein orone which would significantly alter protein expression. Less severedisruptive mutations would include small in-frame deletions andnonconservative base pair substitutions which would have a significanteffect on the protein produced, such as changes to or from a cysteineresidue, from a basic to an acidic amino acid or vice versa, from ahydrophobic to hydrophilic amino acid or vice versa, or other mutationswhich would affect secondary, tertiary or quaternary protein structure.Silent mutations or those resulting in conservative amino acidsubstitutions would not generally be expected to disrupt proteinfunction.

According to the diagnostic and prognostic method of the presentinvention, alteration of the wild-type BRCA1 locus is detected. Inaddition, the method can be performed by detecting the wild-type BRCA1locus and confirming the lack of a predisposition to cancer at the BRCA1locus. "Alteration of a wild-type gene" encompasses all forms ofmutations including deletions, insertions and point mutations in thecoding and noncoding regions. Deletions may be of the entire gene or ofonly a portion of the gene. Point mutations may result in stop codons,frameshift mutations or amino acid substitutions. Somatic mutations arethose which occur only in certain tissues, e.g., in the tumor tissue,and are not inherited in the germline. Germline mutations can be foundin any of a body's tissues and are inherited. If only a single allele issomatically mutated, an early neoplastic state is indicated. However, ifboth alleles are somatically mutated, then a late neoplastic state isindicated. The finding of BRCA1 mutations thus provides both diagnosticand prognostic information. A BRCA1 allele which is not deleted (e.g.,found on the sister chromosome to a chromosome carrying a BRCA1deletion) can be screened for other mutations, such as insertions, smalldeletions, and point mutations. It is believed that many mutations foundin tumor tissues will be those leading to decreased expression of theBRCA1 gene product. However, mutations leading to non-functional geneproducts would also lead to a cancerous state. Point mutational eventsmay occur in regulatory regions, such as in the promoter of the gene,leading to loss or diminution of expression of the mRNA. Point mutationsmay also abolish proper RNA processing, leading to loss of expression ofthe BRCA1 gene product, or to a decrease in mRNA stability ortranslation efficiency.

Useful diagnostic techniques include, but are not limited to fluorescentin situ hybridization (FISH), direct DNA sequencing, PFGE analysis,Southern blot analysis, single stranded conformation analysis (SSCA),RNase protection assay, allele-specific oligonucleotide (ASO), dot blotanalysis and PCR-SSCP, as discussed in detail further below.

Predisposition to cancers, such as breast and ovarian cancer, and theother cancers identified herein, can be ascertained by testing anytissue of a human for mutations of the BRCA1 gene. For example, a personwho has inherited a germline BRCA1 mutation would be prone to developcancers. This can be determined by testing DNA from any tissue of theperson's body. Most simply, blood can be drawn and DNA extracted fromthe cells of the blood. In addition, prenatal diagnosis can beaccomplished by testing fetal cells, placental cells or amniotic cellsfor mutations of the BRCA1 gene. Alteration of a wild-type BRCA1 allele,whether, for example, by point mutation or deletion, can be detected byany of the means discussed herein.

There are several methods that can be used to detect DNA sequencevariation. Direct DNA sequencing, either manual sequencing or automatedfluorescent sequencing can detect sequence variation. For a gene aslarge as BRCA1, manual sequencing is very labor-intensive, but underoptimal conditions, mutations in the coding sequence of a gene arerarely missed. Another approach is the single-stranded conformationpolymorphism assay (SSCA) (Orita et al., 1989). This method does notdetect all sequence changes, especially if the DNA fragment size isgreater than 200 bp, but can be optimized to detect most DNA sequencevariation. The reduced detection sensitivity is a disadvantage, but theincreased throughput possible with SSCA makes it an attractive, viablealternative to direct sequencing for mutation detection on a researchbasis. The fragments which have shifted mobility on SSCA gels are thensequenced to determine the exact nature of the DNA sequence variation.Other approaches based on the detection of mismatches between the twocomplementary DNA strands include clamped denaturing gel electrophoresis(CDGE) (Sheffield et al., 1991), heteroduplex analysis (HA) (White etal., 1992) and chemical mismatch cleavage (CMC) (Grompe et al., 1989).None of the methods described above will detect large deletions,duplications or insertions, nor will they detect a regulatory mutationwhich affects transcription or translation of the protein. Other methodswhich might detect these classes of mutations such as a proteintruncation assay or the asymmetric assay, detect only specific types ofmutations and would not detect missense mutations. A review of currentlyavailable methods of detecting DNA sequence variation can be found in arecent review by Grompe (1993). Once a mutation is known, an allelespecific detection approach such as allele specific oligonucleotide(ASO) hybridization can be utilized to rapidly screen large numbers ofother samples for that same mutation.

In order to detect the alteration of the wild-type BRCA1 gene in atissue, it is helpful to isolate the tissue free from surrounding normaltissues. Means for enriching tissue preparation for tumor cells areknown in the art. For example, the tissue may be isolated from paraffinor cryostat sections. Cancer cells may also be separated from normalcells by flow cytometry. These techniques, as well as other techniquesfor separating tumor cells from normal cells, are well known in the art.If the tumor tissue is highly contaminated with normal cells, detectionof mutations is more difficult.

A rapid preliminary analysis to detect polymorphisms in DNA sequencescan be performed by looking at a series of Southern blots of DNA cutwith one or more restriction enzymes, preferably with a large number ofrestriction enzymes. Each blot contains a series of normal individualsand a series of cancer cases, tumors, or both. Southern blots displayinghybridizing fragments (differing in length from control DNA when probedwith sequences near or including the BRCA1 locus) indicate a possiblemutation. If restriction enzymes which produce very large restrictionfragments are used, then pulsed field gel electrophoresis (PFGE) isemployed.

Detection of point mutations may be accomplished by molecular cloning ofthe BRCA1 allele(s) and sequencing the allele(s) using techniques wellknown in the art. Alternatively, the gene sequences can be amplifieddirectly from a genomic DNA preparation from the tumor tissue, usingknown techniques. The DNA sequence of the amplified sequences can thenbe determined.

There are six well known methods for a more complete, yet stillindirect, test for confirming the presence of a susceptibilityallele: 1) single stranded conformation analysis (SSCA) (Orita et al.,1989); 2) denaturing gradient gel electrophoresis (DGGE) (Wartell etal., 1990; Sheffield et al., 1989); 3) RNase protection assays(Finkelstein et al., 1990; Kinszler et al., 1991); 4) allele-specificoligonuecleotides (ASOs) (Conner et al., 1983); 5) the use of proteinswhich recognize nucleotide mismatches, such as the E. coli mutS protein(Modrich, 1991); and 6) allele-specific PCR (Rano & Kidd, 1989). Forallele-specific PCR, primers are used which hybridize at their 3' endsto a particular BRCA1 mutation. If the particular BRCA1 mutation is notpresent, an amplification product is not observed. AmplificationRefractory Mutation System (ARMS) can also be used, as disclosed inEuropean Patent Application Publication No. 0332435 and in Newton etal., 1989. Insertions and deletions of genes can also be detected bycloning, sequencing and amplification. In addition, restriction fragmentlength polymorphism (RFLP) probes for the gene or surrounding markergenes can be used to score alteration of an allele or an insertion in apolymorphic fragment. Such a method is particularly useful for screeningrelatives of an affected individual for the presence of the BRCA1mutation found in that individual. Other techniques for detectinginsertions and deletions as known in the art can be used.

In the first three methods (SSCA, DGGE and RNase protection assay), anew electrophoretic band appears. SSCA detects a band which migratesdifferentially because the sequence change causes a difference insingle-strand, intramolecular base pairing. RNase protection involvescleavage of the mutant polynucleotide into two or more smallerfragments. DGGE detects differences in migration rates of mutantsequences compared to wild-type sequences, using a denaturing gradientgel. In an allele-specific oligonucleotide assay, an oligonucleotide isdesigned which detects a specific sequence, and the assay is performedby detecting the presence or absence of a hybridization signal. In themutS assay, the protein binds only to sequences that contain anucleotide mismatch in a heteroduplex between mutant and wild-typesequences.

Mismatches, according to the present invention, are hybridized nucleicacid duplexes in which the two strands are not 100% complementary. Lackof total homology may be due to deletions, insertions, inversions orsubstitutions. Mismatch detection can be used to detect point mutationsin the gene or in its mRNA product. While these techniques are lesssensitive than sequencing, they are simpler to perform on a large numberof tumor samples. An example of a mismatch cleavage technique is theRNase protection method. In the practice of the present invention, themethod involves the use of a labeled riboprobe which is complementary tothe human wild-type BRCA1 gene coding sequence. The riboprobe and eithermRNA or DNA isolated from the tumor tissue are annealed (hybridized)together and subsequently digested with the enzyme RNase A which is ableto detect some mismatches in a duplex RNA structure. If a mismatch isdetected by RNase A, it cleaves at the site of the mismatch. Thus, whenthe annealed RNA preparation is separated on an electrophoretic gelmatrix, if a mismatch has been detected and cleaved by RNase A, an RNAproduct will be seen which is smaller than the full length duplex RNAfor the riboprobe and the mRNA or DNA. The riboprobe need not be thefull length of the BRCA1 mRNA or gene but can be a segment of either. Ifthe riboprobe comprises only a segment of the BRCA1 mRNA or gene, itwill be desirable to use a number of these probes to screen the wholemRNA sequence for mismatches.

In similar fashion, DNA probes can be used to detect mismatches, throughenzymatic or chemical cleavage. See, e.g., Cotton et al., 1988; Shenk etal., 1975; Novack et al., 1986. Alternatively, mismatches can bedetected by shifts in the electrophoretic mobility of mismatchedduplexes relative to matched duplexes. See, e.g., Cariello, 1988. Witheither riboprobes or DNA probes, the cellular mRNA or DNA which mightcontain a mutation can be amplified using PCR (see below) beforehybridization. Changes in DNA of the BRCA1 gene can also be detectedusing Southern hybridization, especially if the changes are grossrearrangements, such as deletions and insertions.

DNA sequences of the BRCA1 gene which have been amplified by use of PCRmay also be screened using allele-specific probes. These probes arenucleic acid oligomers, each of which contains a region of the BRCA1gene sequence harboring a known mutation. For example, one oligomer maybe about 30 nucleotides in length, corresponding to a portion of theBRCA1 gene sequence. By use of a battery of such allele-specific probes,PCR amplification products can be screened to identify the presence of apreviously identified mutation in the BRCA1 gene. Hybridization ofallele-specific probes with amplified BRCA1 sequences can be performed,for example, on a nylon filter. Hybridization to a particular probeunder stringent hybridization conditions indicates the presence of thesame mutation in the tumor tissue as in the allele-specific probe.

The most definitive test for mutations in a candidate locus is todirectly compare genomic BRCA1 sequences from cancer patients with thosefrom a control population. Alternatively, one could sequence messengerRNA after amplification, e.g., by PCR, thereby eliminating the necessityof determining the exon structure of the candidate gene.

Mutations from cancer patients falling outside the coding region ofBRCA1 can be detected by examining the non-coding regions, such asintrons and regulatory sequences near or within the BRCA1 gene. An earlyindication that mutations in noncoding regions are important may comefrom Northern blot experiments that reveal messenger RNA molecules ofabnormal size or abundance in cancer patients as compared to controlindividuals.

Alteration of BRCA1 mRNA expression can be detected by any techniquesknown in the art. These include Northern blot analysis, PCRamplification and RNase protection. Diminished mRNA expression indicatesan alteration of the wild-type BRCA1 gene. Alteration of wild-type BRCA1genes can also be detected by screening for alteration of wild-typeBRCA1 protein. For example, monoclonal antibodies immunoreactive withBRCA1 can be used to screen a tissue. Lack of cognate antigen wouldindicate a BRCA1 mutation. Antibodies specific for products of mutantalleles could also be used to detect mutant BRCA1 gene product. Suchimmunological assays can be done in any convenient formats known in theart. These include Western blots, immunohistochemical assays and ELISAassays. Any means for detecting an altered BRCA1 protein can be used todetect alteration of wild-type BRCA1 genes. Functional assays, such asprotein binding determinations, can be used. In addition, assays can beused which detect BRCA1 biochemical function. Finding a mutant BRCA1gene product indicates alteration of a wild-type BRCA1 gene.

Mutant BRCA1 genes or gene products can also be detected in other humanbody samples, such as serum, stool, urine and sputum. The sametechniques discussed above for detection of mutant BRCA1 genes or geneproducts in tissues can be applied to other body samples. Cancer cellsare sloughed off from tumors and appear in such body samples. Inaddition, the BRCA1 gene product itself may be secreted into theextracellular space and found in these body samples even in the absenceof cancer cells. By screening such body samples, a simple earlydiagnosis can be achieved for many types of cancers. In addition, theprogress of chemotherapy or radiotherapy can be monitored more easily bytesting such body samples for mutant BRCA1 genes or gene products.

The methods of diagnosis of the present invention are applicable to anytumor in which BRCA1 has a role in tumorigenesis. The diagnostic methodof the present invention is useful for clinicians, so they can decideupon an appropriate course of treatment.

The primer pairs of the present invention are useful for determinationof the nucleotide sequence of a particular BRCA1 allele using PCR. Thepairs of single-stranded DNA primers can be annealed to sequences withinor surrounding the BRCA1 gene on chromosome 17q21 in order to primeamplifying DNA synthesis of the BRCA1 gene itself. A complete set ofthese primers allows synthesis of all of the nucleotides of the BRCA1gene coding sequences, i.e., the exons. The set of primers preferablyallows synthesis of both intron and exon sequences. Allele-specificprimers can also be used. Such primers anneal only to particular BRCA1mutant alleles, and thus will only amplify a product in the presence ofthe mutant allele as a template.

In order to facilitate subsequent cloning of amplified sequences,primers may have restriction enzyme site sequences appended to their 5'ends. Thus, all nucleotides of the primers are derived from BRCA1sequences or sequences adjacent to BRCA1, except for the few nucleotidesnecessary to form a restriction enzyme site. Such enzymes and sites arewell known in the art. The primers themselves can be synthesized usingtechniques which are well known in the art. Generally, the primers canbe made using oligonucleotide synthesizing machines which arecommercially available. Given the sequence of the BRCA1 open readingframe shown in SEQ ID NO: 1, design of particular primers is well withinthe skill of the art.

The nucleic acid probes provided by the present invention are useful fora number of purposes. They can be used in Southern hybridization togenomic DNA and in the RNase protection method for detecting pointmutations already discussed above. The probes can be used to detect PCRamplification products. They may also be used to detect mismatches withthe BRCA1 gene or mRNA using other techniques.

It has been discovered that individuals with the wild-type BRCA1 gene donot have cancer which results from the BRCA1 allele. However, mutationswhich interfere with the function of the BRCA1 protein are involved inthe pathogenesis of cancer. Thus, the presence of an altered (or amutant) BRCA1 gene which produces a protein having a loss of function,or altered function, directly correlates to an increased risk of cancer.In order to detect a BRCA1 gene mutation, a biological sample isprepared and analyzed for a difference between the sequence of the BRCA1allele being analyzed and the sequence of the wild-type BRCA1 allele.Mutant BRCA1 alleles can be initially identified by any of thetechniques described above. The mutant alleles are then sequenced toidentify the specific mutation of the particular mutant allele.Alternatively, mutant BRCA1 alleles can be initially identified byidentifying mutant (altered) BRCA1 proteins, using conventionaltechniques. The mutant alleles are then sequenced to identify thespecific mutation for each allele. The mutations, especially those whichlead to an altered function of the BRCA1 protein, are then used for thediagnostic and prognostic methods of the present invention.

DEFINITIONS

The present invention employs the following definitions:

"Amplification of Polynucleotides" utilizes methods such as thepolymerase chain reaction (PCR), ligation amplification (or ligase chainreaction, LCR) and amplification methods based on the use of Q-betareplicase. These methods are well known and widely practiced in the art.See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et al., 1990(for PCR); and Wu et al., 1989a (for LCR). Reagents and hardware forconducting PCR are commercially available. Primers useful to amplifysequences from the BRCA1 region are preferably complementary to, andhybridize specifically to sequences in the BRCA1 region or in regionsthat flank a target region therein. BRCA1 sequences generated byamplification may be sequenced directly. Alternatively, but lessdesirably, the amplified sequence(s) may be cloned prior to sequenceanalysis. A method for the direct cloning and sequence analysis ofenzymatically amplified genomic segments has been described by Scharf,1986.

"Analyte polynucleotide" and "analyte strand" refer to a single- ordouble-stranded polynucleotide which is suspected of containing a targetsequence, and which may be present in a variety of types of samples,including biological samples.

"Antibodies." The present invention also provides polyclonal and/ormonoclonal antibodies and fragments thereof, and immunologic bindingequivalents thereof, which are capable of specifically binding to theBRCA1 polypeptides and fragments thereof or to polynucleotide sequencesfrom the BRCA1 region, particularly from the BRCA1 locus or a portionthereof. The term "antibody" is used both to refer to a homogeneousmolecular entity, or a mixture such as a serum product made up of aplurality of different molecular entities. Polypeptides may be preparedsynthetically in a peptide synthesizer and coupled to a carrier molecule(e.g., keyhole limpet hemocyanin) and injected over several months intorabbits. Rabbit sera is tested for immunoreactivity to the BRCA1polypeptide or fragment. Monoclonal antibodies may be made by injectingmice with the protein polypeptides, fusion proteins or fragmentsthereof. Monoclonal antibodies will be screened by ELISA and tested forspecific immunoreactivity with BRCA1 polypeptide or fragments thereof.See, Harlow & Lane, 1988. These antibodies will be useful in assays aswell as pharmaceuticals.

Once a sufficient quantity of desired polypeptide has been obtained, itmay be used for various purposes. A typical use is the production ofantibodies specific for binding. These antibodies may be eitherpolyclonal or monoclonal, and may be produced by in vitro or in vivotechinques well known in the art. For production of polyclonalantibodies, an appropriate target immune system, typically mouse orrabbit, is selected. Substantially purified antigen is presented to theimmune system in a fashion determined by methods appropriate for theanimal and by other parameters well known to immunologists. Typicalsites for injection are in footpads, intramuscularly, intraperitoneally,or intradermally. Of course, other species may be substituted for mouseor rabbit. Polyclonal antibodies are then purified using techniquesknown in the art, adjusted for the desired specificity.

An immunological response is usually assayed with an immunoassay.Normally, such immunoassays involve some purification of a source ofantigen, for example, that produced by the same cells and in the samefashion as the antigen. A variety of immunoassay methods are well knownin the art. See, e.g., Harlow & Lane, 1988, or Goding, 1986.

Monoclonal antibodies with affinities of 10⁻⁸ M⁻¹ or preferably 10⁻⁹ to10⁻¹⁰ M⁻¹ or stronger will typically be made by standard procedures asdescribed, e.g., in Harlow & Lane, 1988 or Goding, 1986. Briefly,appropriate animals will be selected and the desired immunizationprotocol followed. After the appropriate period of time, the spleens ofsuch animals are excised and individual spleen cells fused, typically,to immortalized myeloma cells under appropriate selection conditions.Thereafter, the cells are clonally separated and the supernatants ofeach clone tested for their production of an appropriate antibodyspecific for the desired region of the antigen.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides, or alternatively, to selection of librariesof antibodies in phage or similar vectors. See Huse et al., 1989. Thepolypeptides and antibodies of the present invention may be used with orwithout modification. Frequently, polypeptides and antibodies will belabeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentagents, chemiluminescent agents, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241. Also, recombinant immunoglobulins may be produced (see U.S.Pat. No. 4,816,567).

"Binding partner" refers to a molecule capable of binding a ligandmolecule with high specificity, as for example, an antigen and anantigen-specific antibody or an enzyme and its inhibitor. In general,the specific binding partners must bind with sufficient affinity toimmobilize the analyte copy/complementary strand duplex (in the case ofpolynucleotide hybridization) under the isolation conditions. Specificbinding partners are known in the art and include, for example, biotinand avidin or streptavidin, IgG and protein A, the numerous, knownreceptor-ligand couples, and complementary polynucleotide strands. Inthe case of complementary polynucleotide binding partners, the partnersare normally at least about 15 bases in length, and may be at least 40bases in length. The polynucleotides may be composed of DNA, RNA, orsynthetic nucleotide analogs.

A "biological sample" refers to a sample of tissue or fluid suspected ofcontaining an analyte polynucleotide or polypeptide from an individualincluding, but not limited to, e.g., plasma, serum, spinal fluid, lymphfluid, the external sections of the skin, respiratory, intestinal, andgenitourinary tracts, tears, saliva, blood cells, tumors, organs, tissueand samples of in vitro cell culture constituents.

As used herein, the terms "diagnosing" or "prognosing," as used in thecontext of neoplasia, are used to indicate 1) the classification oflesions as neoplasia, 2) the determination of the severity of theneoplasia, or 3) the monitoring of the disease progression, prior to,during and after treatment.

"Encode". A polynucleotide is said to "encode" a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor and/or the polypeptide or a fragment thereof. The anti-sense strandis the complement of such a nucleic acid, and the encoding sequence canbe deduced therefrom.

"Isolated" or "substantially pure". An "isolated" or "substantiallypure" nucleic acid (e.g., an RNA, DNA or a mixed polymer) is one whichis substantially separated from other cellular components whichnaturally accompany a native human sequence or protein, e.g., ribosomes,polymerases, many other human genome sequences and proteins. The termembraces a nucleic acid sequence or protein which has been removed fromits naturally occurring environment, and includes recombinant or clonedDNA isolates and chemically synthesized analogs or analogs biologicallysynthesized by heterologous systems.

"BRCA1 Allele" refers to normal alleles of the BRCA1 locus as well asalleles carrying variations that predispose individuals to developcancer of many sites including, for example, breast, ovarian, colorectaland prostate cancer. Such predisposing alleles are also called "BRCA1susceptibility alleles".

"BRCA1 Locus," "BRCA1 Gene," "BRCA1 Nucleic Acids" or "BRCA1Polynucleotide" each refer to polynucleotides, all of which are in theBRCA1 region, that are likely to be expressed in normal tissue, certainalleles of which predispose an individual to develop breast, ovarian,colorectal and prostate cancers. Mutations at the BRCA1 locus may beinvolved in the initiation and/or progression of other types of tumors.The locus is indicated in part by mutations that predispose individualsto develop cancer. These mutations fall within the BRCA1 regiondescribed infra. The BRCA1 locus is intended to include codingsequences, intervening sequences and regulatory elements controllingtranscription and/or translation. The BRCA1 locus is intended to includeall allelic variations of the DNA sequence.

These terms, when applied to a nucleic acid, refer to a nucleic acidwhich encodes a BRCA1 polypeptide, fragment, homolog or variant,including, e.g., protein fusions or deletions. The nucleic acids of thepresent invention will possess a sequence which is either derived from,or substantially similar to a natural BRCA1-encoding gene or one havingsubstantial homology with a natural BRCA1-encoding gene or a portionthereof. The coding sequence for a BRCA1 polypeptide is shown in SEQ IDNO:1, with the amino acid sequence shown in SEQ ID NO:2.

The polynucleotide compositions of this invention include RNA, cDNA,genomic DNA, synthetic forms, and mixed polymers, both sense andantisense strands, and may be chemically or biochemically modified ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those skilled in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

The present invention provides recombinant nucleic acids comprising allor part of the BRCA1 region. The recombinant construct may be capable ofreplicating autonomously in a host cell. Alternatively, the recombinantconstruct may become integrated into the chromosomal DNA of the hostcell. Such a recombinant polynucleotide comprises a polynucleotide ofgenomic, cDNA, semi-synthetic, or synthetic origin which, by virtue ofits origin or manipulation, 1) is not associated with all or a portionof a polynucleotide with which it is associated in nature; 2) is linkedto a polynucleotide other than that to which it is linked in nature; or3) does not occur in nature.

Therefore, recombinant nucleic acids comprising sequences otherwise notnaturally occurring are provided by this invention. Although thewild-type sequence may be employed, it will often be altered, e.g., bydeletion, substitution or insertion.

cDNA or genomic libraries of various types may be screened as naturalsources of the nucleic acids of the present invention, or such nucleicacids may be provided by amplification of sequences resident in genomicDNA or other natural sources, e.g., by PCR. The choice of cDNA librariesnormally corresponds to a tissue source which is abundant in mRNA forthe desired proteins. Phage libraries are normally preferred, but othertypes of libraries may be used. Clones of a library are spread ontoplates, transferred to a substrate for screening, denatured and probedfor the presence of desired sequences.

The DNA sequences used in this invention will usually comprise at leastabout five codons (15 nucleotides), more usually at least about 7-15codons, and most preferably, at least about 35 codons. One or moreintrons may also be present. This number of nucleotides is usually aboutthe minimal length required for a successful probe that would hybridizespecifically with a BRCA1-encoding sequence.

Techniques for nucleic acid manipulation are described generally, forexample, in Sambrook et al., 1989 or Ausubel et al., 1992. Reagentsuseful in applying such techniques, such as restriction enzymes and thelike, are widely known in the art and commercially available from suchvendors as New England BioLabs, Boehringer Mannheim, Amersham, PromegaBiotec, U. S. Biochemicals, New England Nuclear, and a number of othersources. The recombinant nucleic acid sequences used to produce fusionproteins of the present invention may be derived from natural orsynthetic sequences. Many natural gene sequences are obtainable fromvarious cDNA or from genomic libraries using appropriate probes. See,GenBank, National Institutes of Health.

"BRCA1 Region" refers to a portion of human chromosome 17q21 bounded bythe markers tdj1474 and U5R. This region contains the BRCA1 locus,including the BRCA1 gene.

As used herein, the terms "BRCA1 locus," "BRCA1 allele" and "BRCA1region" all refer to the double-stranded DNA comprising the locus,allele, or region, as well as either of the single-stranded DNAscomprising the locus, allele or region.

As used herein, a "portion" of the BRCA1 locus or region or allele isdefined as having a minimal size of at least about eight nucleotides, orpreferably about 15 nucleotides, or more preferably at least about 25nucleotides, and may have a minimal size of at least about 40nucleotides.

"BRCA1 protein" or "BRCA1 polypeptide" refer to a protein or polypeptideencoded by the BRCA1 locus, variants or fragments thereof. The term"polypeptide" refers to a polymer of amino acids and its equivalent anddoes not refer to a specific length of the product; thus, peptides,oligopeptides and proteins are included within the definition of apolypeptide. This term also does not refer to, or exclude modificationsof the polypeptide, for example, glycosylations, acetylations,phosphorylations, and the like. Included within the definition are, forexample, polypeptides containing one or more analogs of an amino acid(including, for example, unnatural amino acids, etc.), polypeptides withsubstituted linkages as well as other modifications known in the art,both naturally and non-naturally occurring. Ordinarily, suchpolypeptides will be at least about 50% homologous to the native BRCA1sequence, preferably in excess of about 90%, and more preferably atleast about 95% homologous. Also included are proteins encoded by DNAwhich hybridize under high or low stringency conditions, toBRCA1-encoding nucleic acids and closely related polypeptides orproteins retrieved by antisera to the BRCA1 protein(s).

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acids, usually at least about 20 residues,more usually at least about 24 residues, typically at least about 28residues, and preferably more than about 35 residues.

"Operably linked" refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended mainer. For instance, a promoter is operably linked to a codingsequence if the promoter affects its transcription or expression.

"Probes". Polynucleotide polymorphisms associated with BRCA1 alleleswhich predispose to certain cancers or are associated with most cancersare detected by hybridization with a polynucleotide probe which forms astable hybrid with that of the target sequence, under stringent tomoderately stringent hybridization and wash conditions. If it isexpected that the probes will be perfectly complementary to the targetsequence, stringent conditions will be used. Hybridization stringencymay be lessened if some mismatching is expected, for example, ifvariants are expected with the result that the probe will not becompletely complementary. Conditions are chosen which rule outnonspecific/adventitious bindings, that is, which minimize noise. Sincesuch indications identify neutral DNA polymorphisms as well asmutations, these indications need further analysis to demonstratedetection of a BRCA1 susceptibility allele.

Probes for BRCA1 alleles may be derived from the sequences of the BRCA1region or its cDNAs. The probes may be of any suitable length, whichspan all or a portion of the BRCA1 region, and which allow specifichybridization to the BRCA1 region. If the target sequence contains asequence identical to that of the probe, the probes may be short, e.g.,in the range of about 8-30 base pairs, since the hybrid will berelatively stable under even stringent conditions. If some degree ofmismatch is expected with the probe, i.e., if it is suspected that theprobe will hybridize to a variant region, a longer probe may be employedwhich hybridizes to the target sequence with the requisite specificity.

The probes will include an isolated polynucleotide attached to a labelor reporter molecule and may be used to isolate other polynucleotidesequences, having sequence similarity by standard methods. Fortechniques for preparing and labeling probes see, e.g., Sambrook et al.,1989 or Ausubel et al., 1992. Other similar polynucleotides may beselected by using homologous polynucleotides. Alternatively,polynucleotides encoding these or similar polypeptides may besynthesized or selected by use of the redundancy in the genetic code.Various codon substitutions may be introduced, e.g., by silent changes(thereby producing various restriction sites) or to optimize expressionfor a particular system. Mutations may be introduced to modify theproperties of the polypeptide, perhaps to change ligand-bindingaffinities, interchain affinities, or the polypeptide degradation orturnover rate.

Probes comprising synthetic oligonucleotides or other polynucleotides ofthe present invention may be derived from naturally occurring orrecombinant single- or double-stranded polynucleotides, or be chemicallysynthesized. Probes may also be labeled by nick translation, Klenowfill-in reaction, or other methods known in the art.

Portions of the polynucleotide sequence having at least about eightnucleotides, usually at least about 15 nucleotides, and fewer than about6 kb, usually fewer than about 1.0 kb, from a polynucleotide sequenceencoding BRCA1 are preferred as probes. The probes may also be used todetermine whether mRNA encoding BRCA1 is present in a cell or tissue.

"Protein modifications or fragments" are provided by the presentinvention for BRCA1 poly-peptides or fragments thereof which aresubstantially homologous to primary structural sequence but whichinclude, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate unusual amino acids. Suchmodifications include, for example, acetylation, carboxylation,phosphoiylation, glycosylation, ubiquitination, labeling, e.g., withradionuclides, and various enzymatic modifications, as will be readilyappreciated by those well skilled in the art. A variety of methods forlabeling polypeptides and of substituents or labels useful for suchpurposes are well known in the art, and include radioactive isotopessuch as ³² P, ligands which bind to labeled antiligands (e.g.,antibodies), fluorophores, chemiluminescent agents, enzymes, andantiligands which can serve as specific binding pair members for alabeled ligand. The choice of label depends on the sensitivity required,ease of conjugation with the primer, stability requirements, andavailable instrumentation. Methods of labeling polypeptides are wellknown in the art. See, e.g., Sambrook et al., 1989 or Ausubel et al.,1992.

Besides substantially full-length polypeptides, the present inventionprovides for biologically active fragments of the polypeptides.Significant biological activities include ligand-binding, immunologicalactivity and other biological activities characteristic of BRCA1polypeptides. Immunological activities include both immunogenic functionin a target immune system, as well as sharing of immunological epitopesfor binding, serving as either a competitor or substitute antigen for anepitope of the BRCA1 protein. As used herein, "epitope" refers to anantigenic determinant of a polypeptide. An epitope could comprise threeamino acids in a spatial conformation which is unique to the epitope.Generally, an epitope consists of at least five such amino acids, andmore usually consists of at least 8-10 such amino acids. Methods ofdetermining the spatial conformation of such amino acids are known inthe art.

For immunological purposes, tandem-repeat polypeptide segments may beused as immunogens, thereby producing highly antigenic proteins.Alternatively, such polypeptides will serve as highly efficientcompetitors for specific binding. Production of antibodies specific forBRCA1 polypeptides or fragments thereof is described below.

The present invention also provides for fusion polypeptides, comprisingBRCA1 polypeptides and fragments. Homologous polypeptides may be fusionsbetween two or more BRCA1 polypeptide sequences or between the sequencesof BRCA1 and a related protein. Likewise, heterologous fusions may beconstructed which would exhibit a combination of properties oractivities of the derivative proteins. For example, ligand-binding orother domains may be "swapped" between different new fusion polypeptidesor fragments. Such homologous or heterologous fusion polypeptides maydisplay, for example, altered strength or specificity of binding. Fusionpartners include immunoglobulins, bacterial β-galactosidase, trpE,protein A, β-lactamase, alpha amylase, alcohol dehydrogenase and yeastalpha mating factor. See, e.g., Godowski et al., 1988.

Fusion proteins will typically be made by either recombinant nucleicacid methods, as described below, or may be chemically synthesized.Techniques for the synthesis of polypeptides are described, for example,in Merrifield, 1963.

"Protein purification" refers to various methods for the isolation ofthe BRCA1 polypeptides from other biological material, such as fromcells transformed with recombinant nucleic acids encoding BRCA1, and arewell known in the art. For example, such polypeptides may be purified byimmunoaffinity chromatography employing, e.g., the antibodies providedby the present invention. Various methods of protein purification arewell known in the art, and include those described in Deutscher, 1990and Scopes, 1982.

The terms "isolated", "substantially pure", and "substantiallyhomogeneous" are used interchangeably to describe a protein orpolypeptide which has been separated from components which accompany itin its natural state. A monomeric protein is substantially pure when atleast about 60 to 75% of a sample exhibits a single polypeptidesequence. A substantially pure protein will typically comprise about 60to 90% W/W of a protein sample, more usually about 95%, and preferablywill be over about 99% pure. Protein purity or homogeneity may beindicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a protein sample, followed byvisualizing a single polypeptide band upon staining the gel. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art which are utilized for purification.

A BRCA1 protein is substantially free of naturally associated componentswhen it is separated from the native contaminants which accompany it inits natural state. Thus, a polypeptide which is chemically synthesizedor synthesized in a cellular system different from the cell from whichit naturally originates will be substantially free from its naturallyassociated components. A protein may also be rendered substantially freeof naturally associated components by isolation, using proteinpurification techniques well known in the art.

A polypeptide produced as an expression product of an isolated andmanipulated genetic sequence is an "isolated polypcptide," as usedherein, even if expressed in a homologous cell type. Synthetically madeforms or molecules expressed by heterologous cells are inherentlyisolated molecules.

"Recombinant nucleic acid" is a nucleic acid which is not naturallyoccurring, or which is made by the artificial combination of twootherwise separated segments of sequence. This artificial combination isoften accomplished by either chemical synthesis means, or by theartificial manipulation of isolated segments of nucleic acids, e.g., bygenetic engineering techniques. Such is usually done to replace a codonwith a redundant codon encoding the same or a conservative amino acid,while typically introducing or removing a sequence recognition site.Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a desired combination of functions.

"Regulatory sequences" refers to those sequences normally within 100 kbof the coding region of a locus, but they may also be more distant fromthe coding region, which affect the expression of the gene (includingtranscription of the gene, and translation, splicing, stability or thelike of the messenger RNA).

"Substantial homology or similarity". A nucleic acid or fragment thereofis "substantially homologous" ("or substantially similar") to anotherif, when optimally aligned (with appropriate nucleotide insertions ordeletions) with the other nucleic acid (or its complementary strand),there is nucleotide sequence identity in at least about 60% of thenucleotide bases, usually at least about 70%, more usually at leastabout 80%, preferably at least about 90%, and more preferably at leastabout 95-98% of the nucleotide bases.

Alternatively, substantial homology or (similarity) exists when anucleic acid or fragment thereof will hybridize to another nucleic acid(or a complementary strand thereof) under selective hybridizationconditions, to a strand, or to its complement. Selectivity ofhybridization exists when hybridization which is substantially moreselective than total lack of specificity occurs. Typically, selectivehybridization will occur when there is at least about 55% homology overa stretch of at least about 14 nucleotides, preferably at least about65%, more preferably at least about 75%, and most preferably at leastabout 90%. See, Kanehisa, 1984. The length of homology comparison, asdescribed, may be over longer stretches, and in certain embodiments willoften be over a stretch of at least about nine nucleotides, usually atleast about 20 nucleotides, more usually at least about 24 nucleotides,typically at least about 28 nucleotides, more typically at least about32 nucleotides, and preferably at least about 36 or more nucleotides.

Nucleic acid hybridization will be affected by such conditions as saltconcentration, temperature, or organic solvents, in addition to the basecomposition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridizing nucleic acids, aswill be readily appreciated by those skilled in the art. Stringenttemperature conditions will generally include temperatures in excess of30° C. typically in excess of 37° C. and preferably in excess of 45° C.Stringent salt conditions will ordinarily be less than 1000 mM,typically less than 500 mM, and preferably less than 200 mM. However,the combination of parameters is much more important than the measure ofany single parameter. See, e.g., Wetmur & Davidson, 1968.

Probe sequences may also hybridize specifically to duplex DNA undercertain conditions to form triplex or other higher order DNA complexes.The preparation of such probes and suitable hybridization conditions arewell known in the art.

The terms "substantial homology" or "substantial identity", whenreferring to polypeptides, indicate that the polypeptide or protein inquestion exhibits at least about 30% identity with an entirenaturally-occurring protein or a portion thereof, usually at least about70% identity, and preferably at least about 95% identity.

"Substantially similar function" refers to the function of a modifiednucleic acid or a modified protein, with reference to the wild-typeBRCA1 nucleic acid or wild-type BRCA1 polypeptide. The modifiedpolypeptide will be substantially homologous to the wild-type BRCA1polypeptide and will have substantially the same function. The modifiedpolypeptide may have an altered amino acid sequence and/or may containmodified amino acids. In addition to the similarity of function, themodified polypeptide may have other useful properties, such as a longerhalf-life. The similarity of function (activity) of the modifiedpolypeptide may be substantially the same as the activity of thewild-type BRCA1 polypeptide. Alternatively, the similarity of function(activity) of the modified polypeptide may be higher than the activityof the wild-type BRCA1 polypeptide. The modified poly-peptide issynthesized using conventional techniques, or is encoded by a modifiednucleic acid and produced using conventional techniques. The modifiednucleic acid is prepared by conventional techniques. A nucleic acid witha function substantially similar to the wild-type BRCA1 gene functionproduces the modified protein described above.

Homology, for polypeptides, is typically measured using sequenceanalysis software. See, e.g., the Sequence Analysis Software Package ofthe Genetics Computer Group, University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wisc. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions and other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine.

A polypeptide "fragment," "portion" or "segment" is a stretch of aminoacid residues of at least about five to seven contiguous amino acids,often at least about seven to nine contiguous amino acids, typically atleast about nine to 13 contiguous amino acids and, most preferably, atleast about 20 to 30 or more contiguous amino acids.

The polypeptides of the present invention, if soluble, may be coupled toa solid-phase support, e.g., nitrocellulose, nylon, column packingmaterials (e.g., Sepharose beads), magnetic beads, glass wool, plastic,metal, polymer gels, cells, or other substrates. Such supports may takethe form, for example, of beads, wells, dipsticks, or membranes.

"Target region" refers to a region of the nucleic acid which isamplified and/or detected. The term "target sequence" refers to asequence with which a probe or primer will form a stable hybrid underdesired conditions.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, and immunology. See, e.g.,Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al., 1992;Glover, 1985; Anand, 1992; Guthrie & Fink, 1991. A general discussion oftechniques and materials for human gene mapping, including mapping ofhuman chromosome 17q, is provided, e.g., in White and Lalouel, 1988.

Preparation of Recombinant or Chemically Synthesized Nucleic Acids;Vectors, Transformation, Host Cells

Large amounts of the polynucleotides of the present invention may beproduced by replication in a suitable host cell. Natural or syntheticpolynucleotide fragments coding for a desired fragment will beincorporated into recombinant polynucleotide constructs, usually DNAconstructs, capable of introduction into and replication in aprokaryotic or eukaryotic cell. Usually the polynucleotide constructswill be suitable for replication in a unicellular host, such as yeast orbacteria, but may also be intended for introduction to (with and withoutintegration within the genome) cultured mammalian or plant or othereukaryotic cell lines. The purification of nucleic acids produced by themethods of the present invention is described, e.g., in Sambrook et al.,1989 or Ausubel et al., 1992.

The polynucleotides of the present invention may also be produced bychemical synthesis, e.g., by the phosphoramidite method described byBeaucage & Carruthers, 1981 or the triester method according toMatteucci and Caruthers, 1981, and may be performed on commercial,automated oligonucleotide synthesizers. A double-stranded fragment maybe obtained from the single-stranded product of chemical synthesiseither by synthesizing the complementary strand and annealing thestrands together under appropriate conditions or by adding thecomplementary strand using DNA polymerase with an appropriate primersequence.

Polynucleotide constructs prepared for introduction into a prokaryoticor eukaryotic host may comprise a replication system recognized by thehost, including the intended polynucleotide fragment encoding thedesired polypeptide, and will preferably also include transcription andtranslational initiation regulatory sequences operably linked to thepolypeptide encoding segment. Expression vectors may include, forexample, an origin of replication or autonomously replicating sequence(ARS) and expression control sequences, a promoter, an enhancer andnecessary processing information sites, such as ribosome-binding sites,RNA splice sites, polyadenylation sites, transcriptional terminatorsequences, and mRNA stabilizing sequences. Secretion signals may also beincluded where appropriate, whether from a native BRCA1 protein or fromother receptors or from secreted polypeptides of the same or relatedspecies, which allow the protein to cross and/or lodge in cellmembranes, and thus attain its functional topology, or be secreted fromthe cell. Such vectors may be prepared by means of standard recombinanttechniques well known in the art and discussed, for example, in Sambrooket al., 1989 or Ausubel et al. 1992.

An appropriate promoter and other necessary vector sequences will beselected so as to be functional in the host, and may include, whenappropriate, those naturally associated with BRCA1 genes. Examples ofworkable combinations of cell lines and expression vectors are describedin Sambrook et al., 1989 or Ausubel et al., 1992; see also, e.g.,Metzger et al., 1988. Many useful vectors are known in the art and maybe obtained from such vendors as Stratagene, New England Biolabs,Promega Biotech, and others. Promoters such as the trp, lac and phagepromoters, tRNA promoters and glycolytic enzyme promoters may be used inprokaryotic hosts. Useful yeast promoters include promoter regions formetallothionein, 3-phosphoglycerate kinase or other glycolytic enzymessuch as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymesresponsible for maltose and galactose utilization, and others. Vectorsand promoters suitable for use in yeast expression are further describedin Hitzeman et al., EP 73,675A. Appropriate non-native mammalianpromoters might include the early and late promoters from SV40 (Fiers etal., 1978) or promoters derived from murine Moloney leukemia virus,mouse tumor virus, avian sarcoma viruses, adenovirus II, bovinepapilloma virus or polyoma. In addition, the construct may be joined toan amplifiable gene (e.g., DHFR) so that multiple copies of the gene maybe made. For appropriate enhancer and other expression controlsequences, see also Enhancers and Eukaryolic Gene Expression, ColdSpring Harbor Press, Cold Spring Harbor, N. Y. (1983).

While such expression vectors may replicate autonomously, they may alsoreplicate by being inserted into the genome of the host cell, by methodswell known in the art.

Expression and cloning vectors will likely contain a selectable marker,a gene encoding a protein necessary for survival or growth of a hostcell transformed with the vector. The presence of this gene ensuresgrowth of only those host cells which express the inserts. Typicalselection genes encode proteins that a) confer resistance to antibioticsor other toxic substances, e.g. ampicillin, neomycin, methotrexate,etc.; b) complement auxotrophic deficiencies, or c) supply criticalnutrients not available from complex media, e.g., the gene encodingD-alanine racemase for Bacilli. The choice of the proper selectablemarker will depend on the host cell, and appropriate markers fordifferent hosts are well known in the art.

The vectors containing the nucleic acids of interest can be transcribedin vitro, and the resulting RNA introduced into the host cell bywell-known methods, e.g., by injection (see, Kubo et al., 1988), or thevectors can be introduced directly into host cells by methods well knownin the art, which vary depending on the type of cellular host, includingelectroporation; transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; infection (where the vector isan infectious agent, such as a retroviral genome); and other methods.See generally, Sambrook et al., 1989 and Ausubel et al., 1992. Theintroduction of the polynucleotides into the host cell by any methodknown in the art, including, inter alia, those described above, will bereferred to herein as "transformation." The cells into which have beenintroduced nucleic acids described above are meant to also include theprogeny of such cells.

Large quantities of the nucleic acids and polypeptides of the presentinvention may be prepared by expressing the BRCA1 nucleic acids orportions thereof in vectors or other expression vehicles in compatibleprokaryotic or eukaryotic host cells. The most commonly used prokaryotichosts are strains of Escherichia coli, although other prokaryotes, suchas Bacillus subtilis or Pseudomonas may also be used.

Mammalian or other eukaryotic host cells, such as those of yeast,filamentous fungi, plant, insect, or amphibian or avian species, mayalso be useful for production of the proteins of the present invention.Propagation of mammalian cells in culture is per se well known. See,Jakoby and Pastan, 1979. Examples of commonly used mammalian host celllines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, andWI38, BHK, and COS cell lines, although it will be appreciated by theskilled practitioner that other cell lines may be appropriate, e.g., toprovide higher expression, desirable glycosylation patterns, or otherfeatures.

Clones are selected by using markers depending on the mode of the vectorconstruction. The marker may be on the same or a different DNA molecule,preferably the same DNA molecule. In prokaryotic hosts, the transformantmay be selected, e.g., by resistance to ampicillin, tetracycline orother antibiotics. Production of a particular product based ontemperature sensitivity may also serve as an appropriate marker.

Prokaryotic or eukaryotic cells transformed with the polynucleotides ofthe present invention will be useful not only for the production of thenucleic acids and polypeptides of the present invention, but also, forexample, in studying the characteristics of BRCA1 polypeptides.

Antisense polynucleotide sequences are useful in preventing ordiminishing the expression of the BRCA1 locus, as will be appreciated bythose skilled in the art. For example, polynucleotide vectors containingall or a portion of the BRCA1 locus or other sequences from the BRCA1region (particularly those flanking the BRCA1 locus) may be placed underthe control of a promoter in an antisense orientation and introducedinto a cell. Expression of such an antisense construct within a cellwill interfere with BRCA1 transcription and/or translation and/orreplication.

The probes and primers based on the BRCA1 gene sequences disclosedherein are used to identify homologous BRCA1 gene sequences and proteinsin other species. These BRCA1 gene sequences and proteins are used inthe diagnostic/prognostic, therapeutic and drug screening methodsdescribed herein for the species from which they have been isolated.

Methods of Use: Nucleic Acid Diagnosis and Diagnostic Kits

In order to detect the presence of a BRCA1 allele predisposing anindividual to cancer, a biological sample such as blood is prepared andanalyzed for the presence or absence of susceptibility alleles of BRCA1.In order to detect the presence of neoplasia, the progression towardmalignancy of a precursor lesion, or as a prognostic indicator, abiological sample of the lesion is prepared and analyzed for thepresence or absence of mutant alleles of BRCA1. Results of these testsand interpretive information are returned to the health care providerfor communication to the tested individual. Such diagnoses may beperformed by diagnostic laboratories, or, alternatively, diagnostic kitsare manufactured and sold to health care providers or to privateindividuals for self-diagnosis.

Initially, the screening method involves amplification of the relevantBRCA1 sequences. In another preferred embodiment of the invention, thescreening method involves a non-PCR based strategy. Such screeningmethods include two-step label amplification methodologies that are wellknown in the art. Both PCR and non-PCR based screening strategies candetect target sequences with a high level of sensitivity.

The most popular method used today is target amplification. Here, thetarget nucleic acid sequence is amplified with polymerases. Oneparticularly preferred method using polymerase-driven amplification isthe polymerase chain reaction (PCR). The polymerase chain reaction andother polymerase-driven amplification assays can achieve over amillion-fold increase in copy number through the use ofpolymerase-driven amplification cycles. Once amplified, the resultingnucleic acid can be sequenced or used as a substrate for DNA probes.

When the probes are used to detect the presence of the target sequences(for example, in screening for cancer susceptibility), the biologicalsample to be analyzed, such as blood or serum, may be treated, ifdesired, to extract the nucleic acids. The sample nucleic acid may beprepared in various ways to facilitate detection of the target sequence;e.g. denaturation, restriction digestion, electrophoresis or dotblotting. The targeted region of the analyte nucleic acid usually mustbe at least partially single-stranded to form hybrids with the targetingsequence of the probe. If the sequence is naturally single-stranded,denaturation will not be required. However, if the sequence isdouble-stranded, the sequence will probably need to be denatured.Denaturation can be carried out by various techniques known in the art.

Analyte nucleic acid and probe are incubated under conditions whichpromote stable hybrid formation of the target sequence in the probe withthe putative targeted sequence in the analyte. The region of the probeswhich is used to bind to the analyte can be made completelycomplementary to the targeted region of human chromosome 17q. Therefore,high stringency conditions are desirable in order to prevent falsepositives. However, conditions of high stringency are used only if theprobes are complementary to regions of the chromosome which are uniquein the genome. The stringency of hybridization is determined by a numberof factors during hybridization and during the washing procedure,including temperature, ionic strength, base composition, probe length,and concentration of formamide. These factors are outlined in, forexample, Maniatis et al., 1982 and Sambrook et al., 1989. Under certaincircumstances, the formation of higher order hybrids, such as triplexes,quadraplexes, etc., may be desired to provide the means of detectingtarget sequences.

Detection, if any, of the resulting hybrid is usually accomplished bythe use of labeled probes. Alternatively, the probe may be unlabeled,but may be detectable by specific binding with a ligand which islabeled, either directly or indirectly. Suitable labels, and methods forlabeling probes and ligands are known in the art, and include, forexample, radioactive labels which may be incorporated by known methods(e.g., nick translation, random priming or kinasing), biotin,fluorescent groups, chemiluminescent groups (e.g., dioxetanes,particularly triggered dioxetanes), enzymes, antibodies and the like.Variations of this basic scheme are known in the art, and include thosevariations that facilitate separation of the hybrids to be detected fromextraneous materials and/or that amplify the signal from the labeledmoiety. A number of these variations are reviewed in, e.g., Matthews &Kricka, 1988; Landegren et al., 1988; Mittlin, 1989; U.S. Pat. No.4,868,105, and in EPO Publication No. 225,807.

As noted above, non-PCR based screening assays are also contemplated inthis invention. An exemplary non-PCR based procedure is provided inExample 11. This procedure hybridizes a nucleic acid probe (or an analogsuch as a methyl phosphonate backbone replacing the normalphosphodiester), to the low level DNA target. This probe may have anenzyme covalently linked to the probe, such that the covalent linkagedoes not interfere with the specificity of the hybridization. Thisenzyme-probe-conjugate-target nucleic acid complex can then be isolatedaway from the free probe enzyme conjugate and a substrate is added forenzyme detection. Enzymatic activity is observed as a change in colordevelopment or luminescent output resulting in a 10³ -10⁶ increase insensitivity. For an example relating to the preparation ofoligodeoxynucleotide-alkaline phosphatase conjugates and their use ashybridization probes see Jablonski et al., 1986.

Two-step label amplification methodologies are known in the art. Theseassays work on the principle that a small ligand (such as digoxigenin,biotin, or the like) is attached to a nucleic acid probe capable ofspecifically binding BRCA1. Exemplary probes are provided in Table 9 ofthis patent application and additionally include the nucleic acid probecorresponding to nucleotide positions 3631 to 3930 of SEQ ID NO:1.Allele specific probes are also contemplated within the scope of thisexample and exemplary allele specific probes include probes encompassingthe predisposing mutations summarized in Tables 11 and 12 of this patentapplication.

In one example, the small ligand attached to the nucleic acid probe isspecifically recognized by an antibody-enzyme conjugate. In oneembodiment of this example, digoxigenin is attached to the nucleic acidprobe. Hybridization is detected by an antibody-alkaline phosphataseconjugate which turns over a chemiluminescent substrate. For methods forlabeling nucleic acid probes according to this embodiment see Martin etal., 1990. In a second example, the small ligand is recognized by asecond ligand-enzyme conjugate that is capable of specificallycomplexing to the first ligand. A well known embodiment of this exampleis the biotin-avidin type of interactions. For methods for labelingnucleic acid probes and their use in biotin-avidin based assays seeRigby et al., 1977 and Nguyen et al., 1992.

It is also contemplated within the scope of this invention that thenucleic acid probe assays of this invention will employ a cocktail ofnucleic acid probes capable of detecting BRCA1. Thus, in one example todetect the presence of BRCA1 in a cell sample, more than one probecomplementary to BRCA1 is employed and in particular the number ofdifferent probes is alternatively 2, 3, or 5 different nucleic acidprobe sequences. In another example, to detect the presence of mutationsin the BRCA1 gene sequence in a patient, more than one probecomplementary to BRCA1 is employed where the cocktail includes probescapable of binding to the allele-specific mutations identified inpopulations of patients with alterations in BRCA1. In this embodiment,any number of probes can be used, and will preferably include probescorresponding to the major gene mutations identified as predisposing anindividual to breast cancer. Some candidate probes contemplated withinthe scope of the invention include probes that include theallele-specific mutations identified in Tables 11 and 12 and those thathave the BRCA1 regions corresponding to SEQ ID NO:1 both 5' and 3' tothe mutation site.

Methods of Use: Peptide Diagnosis and Diagnostic Kits

The neoplastic condition of lesions can also be detected on the basis ofthe alteration of wild-type BRCA1 polypeptide. Such alterations can bedetermined by sequence analysis in accordance with conventionaltechniques. More preferably, antibodies (polyclonal or monoclonal) areused to detect differences in, or the absence of BRCA1 peptides. Theantibodies may be prepared as discussed above under the heading"Antibodies" and as further shown in Examples 12 and 13. Othertechniques for raising and purifying antibodies are well known in theart and any such techniques may be chosen to achieve the preparationsclaimed in this invention. In a preferred embodiment of the invention,antibodies will immunoprecipitate BRCA1 proteins from solution as wellas react with BRCA1 protein on Western or immunoblots of polyacrylamidegels. In another preferred embodiment, antibodies will detect BRCA1proteins in paraffin or frozen tissue sections, using immunocytochemicaltechniques.

Preferred embodiments relating to methods for detecting BRCA1 or itsmutations include enzyme linked immunosorbent assays (ELISA),radioimmunoassays (RIA), immunoradiometric assays (IRMA) andimmtunloenzymatic assays (IEMA), including sandwich assays usingmonoclonal and/or polyclonal antibodies. Exemplary sandwich assays aredescribed by David et al. in U.S. Pat. Nos. 5 4,376,110 and 4,486,530,hereby incorporated by reference, and exemplified in Example 14.

Methods of Use: Drug Screening

This invention is particularly useful for screening compounds by usingthe BRCA1 polypeptide or binding fragment thereof in any of a variety ofdrug screening techniques.

The BRCA1 polypeptide or fragment employed in such a test may either befree in solution, affixed to a solid support, or borne on a cellsurface. One method of drug screening utilizes eucaryotic or procaryotichost cells which are stably transformed with recombinant polynucleotidesexpressing the polypeptide or fragment, preferably in competitivebinding assays. Such cells, either in viable or fixed form, can be usedfor standard binding assays. One may measure, for example, for theformation of complexes between a BRCA1 polypeptide or fragment and theagent being tested, or examine the degree to which the formation of acomplex between a BRCA1 polypeptide or fragment and a known ligand isinterfered with by the agent being tested.

Thus, the present invention provides methods of screening for drugscomprising contacting such an agent with a BRCA1 polypeptide or fragmentthereof and assaying (i) for the presence of a complex between the agentand the BRCA1 polypeptide or fragment, or (ii) for the presence of acomplex between the BRCA1 polypeptide or fragment and a ligand, bymethods well known in the art. In such competitive binding assays theBRCA1 polypeptide or fragment is typically labeled. Free BRCA1polypeptide or fragment is separated from that present in aprotein:protein complex, and the amount of free (i.e., uncomplexed)label is a measure of the binding of the agent being tested to BRCA1 orits interference with BRCA1:ligand binding, respectively.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the BRCA1 polypeptidesand is described in detail in Geysen, PCT published application WO84/03564, published on Sep. 13, 1984. Briefly stated, large numbers ofdifferent small peptide test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The peptide testcompounds are reacted with BRCA1 polypeptide and washed. Bound BRCA1polypeptide is then detected by methods well known in the art.

Purified BRCA1 can be coated directly onto plates for use in theaforementioned drug screening techniques. However, non-neutralizingantibodies to the polypeptide can be used to capture antibodies toimmobilize the BRCA1 polypeptide on the solid phase.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of specifically bindingthe BRCA1 polypeptide compete with a test compound for binding to theBRCA1 polypeptide or fragments thereof. In this manner, the antibodiescan be used to detect the presence of any peptide which shares one ormore antigenic determinants of the BRCA1 polypeptide.

A further technique for drug screening involves the use of hosteukaryotic cell lines or cells (such as described above) which have anonfunctional BRCA1 gene. These host cell lines or cells are defectiveat the BRCA1 polypeptide level. The host cell lines or cells are grownin the presence of drug compound. The rate of growth of the host cellsis measured to determine if the compound is capable of regulating thegrowth of BRCA1 defective cells.

Methods of Use: Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact (e.g., agonists, antagonists, inhibitors) in orderto fashion drugs which are, for example, more active or stable forms ofthe polypeptide, or which, e.g., enhance or interfere with the functionof a polypeptide in vivo. See, e.g., Hodgson, 1991. In one approach, onefirst determines the three-dimensional structure of a protein ofinterest (e.g., BRCA1 polypeptide) or, for example, of theBRCA1-receptor or ligand complex, by x-ray crystallography, by computermodeling or most typically, by a combination of approaches. Less often,useful information regarding the structure of a polypeptide may begained by modeling based on the structure of homologous proteins. Anexample of rational drug design is the development of HIV proteaseinhibitors (Erickson et al., 1990). In addition, peptides (e.g., BRCA1polypeptide) are analyzed by an alanine scan (Wells, 1991). In thistechnique, an amino acid residue is replaced by Ala, and its effect onthe peptide's activity is determined. Each of the amino acid residues ofthe peptide is analyzed in this manner to determine the importantregions of the peptide.

It is also possible to isolate a target-specific antibody, selected by afunctional assay, and then to solve its crystal structure. In principle,this approach yields a pharmacore upon which subsequent drug design canbe based. It is possible to bypass protein crystallography altogether bygenerating anti-idio-typic antibodies (anti-ids) to a functional,pharmacologically active antibody. As a mirror image of a mirror image,the binding site of the anti-ids would be expected to be an analog ofthe original receptor. The anti-id could then be used to identify andisolate peptides from banks of chemically or biologically produced banksof peptides. Selected peptides would then act as the pharmacore.

Thus, one may design drugs which have, e.g., improved BRCA1 polypeptideactivity or stability or which act as inhibitors, agonists, antagonists,etc. of BRCA1 polypeptide activity. By virtue of the availability ofcloned BRCA1 sequences, sufficient amounts of the BRCA1 polypeptide maybe made available to perform such analytical studies as x-raycrystallography. In addition, the knowledge of the BRCA1 proteinsequence provided herein will guide those employing computer modelingtechniques in place of, or in addition to x-ray crystallography.

Methods of Use: Gene Therapy

According to the present invention, a method is also provided ofsupplying wild-type BRCA1 function to a cell which carries mutant BRCA1alleles. Supplying such a function should suppress neoplastic growth ofthe recipient cells. The wild-type BRCA1 gene or a part of the gene maybe introduced into the cell in a vector such that the gene remainsextrachromosomal. In such a situation, the gene will be expressed by thecell from the extrachromosomal location. If a gene fragment isintroduced and expressed in a cell carrying a mutant BRCA1 allele, thegene fragment should encode a part of the BRCA1 protein which isrequired for non-neoplastic growth of the cell. More preferred is thesituation where the wild-type BRCA1 gene or a part thereof is introducedinto the mutant cell in such a way that it recombines with theendogenous mutant BRCA1 gene present in the cell. Such recombinationrequires a double recombination event which results in the correction ofthe BRCA1 gene mutation. Vectors for introduction of genes both forrecombination and for extrachromosomal maintenance are known in the art,and any suitable vector may be used. Methods for introducing DNA intocells such as electroporation, calcium phosphate co-precipitation andviral transduction are known in the art, and the choice of method iswithin the competence of the routineer. Cells transformed with thewild-type BRCA1 gene can be used as model systems to study cancerremission and drug treatments which promote such remission.

As generally discussed above, the BRCA1 gene or fragment, whereapplicable, may be employed in gene therapy methods in order to increasethe amount of the expression products of such genes in cancer cells.Such gene therapy is particularly appropriate for use in both cancerousand pre-cancerous cells, in which the level of BRCA1 polypeptide isabsent or diminished compared to normal cells. It may also be useful toincrease the level of expression of a given BRCA1 gene even in thosetumor cells in which the mutant gene is expressed at a "normal" level,but the gene product is not fully functional.

Gene therapy would be carried out according to generally acceptedmethods, for example, as described by Friedman, 1991. Cells from apatient's tumor would be first analyzed by the diagnostic methodsdescribed above, to ascertain the production of BRCA1 polypeptide in thetumor cells. A virus or plasmid vector (see further details below),containing a copy of the BRCA1 gene linked to expression controlelements and capable of replicating inside the tumor cells, is prepared.Suitable vectors are known, such as disclosed in U.S. Pat. No. 5,252,479and PCT published application WO 93/07282. The vector is then injectedinto the patient, either locally at the site of the tumor orsystemically (in order to reach any tumor cells that may havemetastasized to other sites). If the transfected gene is not peimanentlyincorporated into the genome of each of the targeted tumor cells, thetreatment may have to be repeated periodically.

Gene transfer systems known in the art may be useful in the practice ofthe gene therapy methods of the present invention. These include viraland nonviral transfer methods. A number of viruses have been used asgene transfer vectors, including papovaviruses, e.g., SV40 (Madzak etal., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988; Gorzigliaand Kapikian, 1992; Quantin et al., 1992; Rosenfeld et al., 1992;Wilkinson et al., 1992; Stratford-Perricaudet et al., 1990), vacciniavirus (Moss, 1992), adenomatus-associated virus (Muzyczka, 1992; Ohi etal., 1990), herpesviruses including HSV and EBV (Margolskee, 1992;Johnson et al., 1992; Fink et al., 1992; Breakfield and Geller, 1987;Freese et al., 1990), and retroviruses of avian (Brandyopadhyay andTemin, 1984; Petropoulos et al., 1992), murine (Miller, 1992; Miller etal., 1985; Sorge et al., 1984; Mann and Baltimore, 1985; Miller et al.,1988), and human origin (Shimada et al., 1991; Helseth et al., 1990;Page et al., 1990; Buchschacher and Panganiban, 1992). Most human genetherapy protocols have been based on disabled murine retroviruses.

Nonviral gene transfer methods known in the art include chemicaltechniques such as calcium phosphate coprecipitation (Graham and van derEb, 1973; Pellicer et al., 1980); mechanical techniques, for examplemicroinjection (Anderson et al., 1980; Gordon et al., 1980; Brinster etal., 1981; Constantini and Lacy, 1981); membrane fusion-mediatedtransfer via liposomes (Felgner et al., 1987; Wang and Huang, 1989;Kaneda et al, 1989; Stewart et al., 1992; Nabel et al., 1990; Lim etal., 1992); and direct DNA uptake and receptor-mediated DNA transfer(Wolff et al., 1990; Wu et al., 1991; Zenke et al., 1990; Wu et al.,1989b; Wolff et al., 1991; Wagner et al., 1990; Wagner et al., 1991;Cotten et al., 1990; Curiel et al., 1991a; Curiel et al., 1991b).Viral-mediated gene transfer can be combined with direct in vivo genetransfer using liposome delivery, allowing one to direct the viralvectors to the tumor cells and not into the surrounding nondividingcells. Alternatively, the retroviral vector producer cell line can beinjected into tumors (Culver et al., 1992). Injection of producer cellswould then provide a continuous source of vector particles. Thistechnique has been approved for use in humans with inoperable braintumors.

In an approach which combines biological and physical gene transfermethods, plasmid DNA of any size is combined with apolylysine-conjugated antibody specific to the adenovirus hexon protein,and the resulting complex is bound to an adenovirus vector. Thetrimolecular complex is then used to infect cells. The adenovirus vectorpermits efficient binding, internalization, and degradation of theendosome before the coupled DNA is damaged.

Liposome/DNA complexes have been shown to be capable of mediating directin vivo gene transfer. While in standard liposome preparations the genetransfer process is nonspecific, localized in vivo uptake and expressionhave been reported in tumor deposits, for example, following direct insitu administration (Nabel, 1992).

Gene transfer techniques which target DNA directly to breast and ovariantissues, e.g., epithelial cells of the breast or ovaries, is preferred.Receptor-mediated gene transfer, for example, is accomplished by theconjugation of DNA (usually in the form of covalently closed supercoiledplasmid) to a protein ligand via polylysine. Ligands are chosen on thebasis of the presence of the corresponding ligand receptors on the cellsurface of the target cell/tissue type. One appropriate receptor/ligandpair may include the estrogen receptor and its ligand, estrogen (andestrogen analogues). These ligand-DNA conjugates can be injecteddirectly into the blood if desired and are directed to the target tissuewhere receptor binding and internalization of the DNA-protein complexoccurs. To overcome the problem of intracellular destruction of DNA,coinfection with adenovirus can be included to disrupt endosomefunction.

The therapy involves two steps which can be performed singly or jointly.In the first step, prepubescent females who carry a BRCA1 susceptibilityallele are treated with a gene delivery vehicle such that some or all oftheir mammary ductal epithelial precursor cells receive at least oneadditional copy of a functional normal BRCA1 allele. In this step, thetreated individuals have reduced risk of breast cancer to the extentthat the effect of the susceptible allele has been countered by thepresence of the normal allele. In the second step of a preventivetherapy, predisposed young females, in particular women who havereceived the proposed gene therapeutic treatment, undergo hormonaltherapy to mimic the effects on the breast of a full term pregnancy.

Methods of Use: Peptide Therapy

Peptides which have BRCA1 activity can be supplied to cells which carrymutant or missing BRCA1 alleles. The sequence of the BRCA1 protein isdisclosed (SEQ ID NO:2). Protein can be produced by expression of thecDNA sequence in bacteria, for example, using known expression vectors.Alternatively, BRCA1 polypeptide can be extracted from BRCA1-producingmammalian cells. In addition, the techniques of synthetic chemistry canbe employed to synthesize BRCA1 protein. Any of such techniques canprovide the preparation of the present invention which comprises theBRCA1 protein. The preparation is substantially free of other humanproteins. This is most readily accomplished by synthesis in amicroorganism or in vitro.

Active BRCA1 molecules can be introduced into cells by microinjection orby use of liposomes, for example. Alternatively, some active moleculesmay be taken up by cells, actively or by diffusion. Extracellularapplication of the BRCA1 gene product may be sufficient to affect tumorgrowth. Supply of molecules with BRCA1 activity should lead to partialreversal of the neoplastic state. Other molecules with BRCA1 activity(for example, peptides, drugs or organic compounds) may also be used toeffect such a reversal. Modified polypeptides having substantiallysimilar function are also used for peptide therapy.

Methods of Use: Transformed Hosts

Similarly, cells and animals which carry a mutant BRCA1 allele can beused as model systems to study and test for substances which havepotential as therapeutic agents. The cells are typically culturedepithelial cells. These may be isolated from individuals with BRCA1mutations, either somatic or germline. Alternatively, the cell line canbe engineered to carry the mutation in the BRCA1 allele, as describedabove. After a test substance is applied to the cells, theneoplastically transformed phenotype of the cell is determined. Anytrait of neoplastically transformed cells can be assessed, includinganchorage-independent growth, tumorigenicity in nude mice, invasivenessof cells, and growth factor dependence. Assays for each of these traitsare known in the art.

Animals for testing therapeutic agents can be selected after mutagenesisof whole animals or after treatment of germline cells or zygotes. Suchtreatments include insertion of mutant BRCA1 alleles, usually from asecond animal species, as well as insertion of disrupted homologousgenes. Alternatively, the endogenous BRCA1 gene(s) of the animals may bedisrupted by insertion or deletion mutation or other genetic alterationsusing conventional techniques (Capecchi, 1989; Valancius and Smithies,1991; Hasty et al., 1991; Shinkai et al., 1992; Mombaerts et al., 1992;Philpott et al., 1992; Snouwaert et al., 1992; Donehower et al., 1992).After test substances have been administered to the animals, the growthof tumors must be assessed. If the test substance prevents or suppressesthe growth of tumors, then the test substance is a candidate therapeuticagent for the treatment of the cancers identified herein. These animalmodels provide an extremely important testing vehicle for potentialtherapeutic products.

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

EXAMPLE 1 Ascertain and Study Kindreds Likely to Have a 17q-LinkedBreast Cancer Susceptibility Locus

Extensive cancer prone kindreds were ascertained by our University ofUtah collaborators from a defined population providing a large set ofextended kindreds with multiple cases of breast cancer and manyrelatives available to study. The large number of meioses present inthese large kindreds provided the power to detect whether the BRCA1locus was segregating, and increased the opportunity for informativerecombinants to occur within the small region being investigated. Thisvastly improved the chances of establishing linkage to the BRCA1 region,and greatly facilitated the reduction of the BRCA1 region to amanageable size, which permits identification of the BRCA1 locus itself.

Each kindred was extended through all available connecting relatives byour collaborators, and to all informative first degree relatives of eachproband or cancer case. For these kindreds, additional breast cancercases and individuals with cancer at other sites of interest (e.g.ovarian) who also appeared in the kindreds were identified through thetumor registry linked files. All breast cancers reported in the kindredwhich were not confirmed in the Utah Cancer Registry were researched.Medical records or death certificates were obtained for confirmation ofall cancers. Each key connecting individual and all informativeindividuals were invited by our collaborators to participate byproviding a blood sample from which DNA was extracted. They also sampledspouses and relatives of deceased cases so that the genotype of thedeceased cases could be inferred from the genotypes of their relatives.

Ten kindreds which had three or more cancer cases with inferablegenotypes were selected for linkage studies to 17q markers from a set of29 kindreds originally ascertained for a study of proliferative breastdisease and breast cancer (Skolnick et al., 1990). The criterion forselection of these kindreds was the presence of two sisters or a motherand her daughter with breast cancer. Additionally, two kindreds whichhave been studied by our collaborators since 1980 as part of theirbreast cancer linkage studies (K1001, K9018), six kindreds ascertainedfor the presence of clusters of breast and/or ovarian cancer (K2019,K2073, K2079, K2080, K2039, K2082) and a self-referred kindred withearly onset breast cancer (K2035) were included. These kindreds wereinvestigated and expanded in our collaborators clinic in the mannerdescribed above. Table 1 displays the characteristics of these 19kindreds which are the subject of subsequent examples. In Table 1, foreach kindred the total number of individuals in our database, the numberof typed individuals, and the minimum, median, and maximum age atdiagnosis of breast/ovarian cancer are reported. Kindreds are sorted inascending order of median age at diagnosis of breast cancer. Four womendiagnosed with both ovarian and breast cancer are counted in bothcategories.

                                      TABLE 1                                     __________________________________________________________________________    Description of the 19 Kindreds                                                No. of       Breast       Ovarian                                             Individuals      Age at Dx    Age at Dx                                       KINDRED                                                                             Total                                                                            Sample                                                                            # Aff.                                                                            Min.                                                                             Med.                                                                             Max.                                                                             # Aff.                                                                            Min.                                                                             Med.                                                                             Max.                                      __________________________________________________________________________    1910  15 10  4   27 34 49 --  -- -- --                                        1001  133                                                                              98  13  28 37 64 --  -- -- --                                        2035  42 25  8   28 37 45  1  -- 60 --                                        2027  21 11  4   34 38 41 --  -- -- --                                        9018  54 17  9   30 40 72  2  46 48 50                                        1925  50 27  4   39 42 53 --  -- -- --                                        1927  49 29  5   32 42 51 --  -- -- --                                        1911  28 21  7   28 42 76 --  -- -- --                                        1929  16 11  4   34 43 73 --  -- -- --                                        1901  35 19  10  31 44 76 --  -- -- --                                        2082  180                                                                              105 20  27 47 67 10  45 52 66                                        2019  42 19  10  42 53 79 --  -- -- --                                        1900  70 23  8   45 55 70  1  -- 78 --                                        2080  264                                                                              74  22+ 27 55 92  4  45 53 71                                        2073  57 29  9   35 57 80 --  -- -- --                                        1917  16  6  4   43 58 61 --  -- -- --                                        1920  22 14  3   62 63 68 --  -- -- --                                        2079  136                                                                              18  14  38 66 84  4  52 59 65                                        2039  87 40  14  44 68 88  4  41 51 75                                        __________________________________________________________________________     +Includes one case of male breast cancer.                                

EXAMPLE 2 Selection of Kindreds Which are Linked to Chromosome 17q andLocalization of BRCA1 to the Interval Mfd15-Mfd188

For each sample collected in these 19 kindreds, DNA was extracted fromblood (or in two cases from paraffin-embedded tissue blocks) usingstandard laboratory protocols. Genotyping in this study was restrictedto short tandem repeat (STR) markers since, in general, they have highheterozygosity and PCR methods offer rapid turnaround while using verysmall amounts of DNA. To aid in this effort, four such STR markers onchromosome 17 were developed by screening a chromosome specific cosmidlibrary for CA positive clones. Three of these markers localized to thelong arm: (46E6, Easton et al., 1993); (42D6, Easton et al., 1993); 26C2(D17S514, Oliphant et al., 1991), while the other, 12G6 (D17S513,Oliphant et al., 1991), localized to the short arm near the p53 tumorsuppressor locus. Two of these, 42D6 and 46E6, were submitted to theBreast Cancer Linkage Consortium for typing of breast cancer families byinvestigators worldwide. Oligonucleotide sequences for markers notdeveloped in our laboratory were obtained from published reports, or aspart of the Breast Cancer Linkage Consortium, or from otherinvestigators. All genotyping films were scored blindly with a standardlane marker used to maintain consistent coding of alleles. Key samplesin the four kindreds presented here underwent duplicate typing for allrelevant markers. All 19 kindreds have been typed for two polymorphic CArepeat markers: 42D6 (D17S588), a CA repeat isolated in our laboratory,and Mfd15 (D17S250), a CA repeat provided by J. Weber (Weber et al.,1990). Several sources of probes were used to create genetic markers onchromosome 17, specifically chromosome 17 cosmid and lambda phagelibraries created from sorted chromosomes by the Los Alamos NationalLaboratories (van Dilla et al., 1986).

LOD scores for each kindred with these two markers (42D6, Mfd15) and athird marker, Mfd188 (D17S579, Hall et al., 1992), located roughlymidway between these two markers, were calculated for two values of therecombination fraction, 0.001 and 0.1. (For calculation of LOD scores,see Oh, 1985). Likelihoods were computed under the model derived byClaus et al., 1991, which assumes an estimated gene frequency of 0.003,a lifetime risk in gene carriers of about 0.80, and population basedage-specific risks for breast cancer in non-gene carriers. Allelefrequencies for the three markers used for the LOD score calculationswere calculated from our own laboratory typings of unrelated individualsin the CEPH panel (White and Lalouel, 1988). Table 2 shows the resultsof the pairwise linkage analysis of each kindred with the three markers42D6, Mfd188, and Mfd15.

                  TABLE 2                                                         ______________________________________                                        Pairwise Linkage Analysis of Kindreds                                                Mfd15         Mfd188        42D6                                              (D17S250)     (D17S579)     (D17S588)                                         Recombination Recombination Recombination                              KINDRED  0.001   0.1     0.001 0.1   0.001 0.1                                ______________________________________                                        1910     0.06    0.30    0.06  0.30  0.06  0.30                               1001     -0.30   -0.09   NT    NT    -0.52 -0.19                              2035     2.34    1.85    0.94  0.90  2.34  1.82                               2027     -1.22   -0.33   -1.20 -0.42 -1.16 -0.33                              9018     -0.54   -0.22   -0.17 -0.10 0.11  0.07                               1925     1.08    0.79    0.55  0.38  -0.11 -0.07                              1927     -0.41   0.01    -0.35 0.07  -0.44 -0.02                              1911     -0.27   -0.13   -0.43 -0.23 0.49  0.38                               1929     -0.49   -0.25   NT    NT    -0.49 -0.25                              1901     1.50    1.17    0.78  0.57  0.65  0.37                               2082     4.25    3.36    6.07  5.11  2.00  3.56                               2019     -0.10   -0.01   -0.11 -0.05 -0.18 -0.10                              1900     -0.14   -0.11   NT    NT    -0.12 -0.05                              2080     -0.16   -0.04   0.76  0.74  -1.25 -0.58                              2073     -0.41   -0.29   0.63  0.49  -0.23 -0.13                              1917     -0.02   -0.02   NT    NT    -0.01 0.00                               1920     -0.03   -0.02   NT    NT    0.00  0.00                               2079     0.02    0.01    -0.01 -0.01 0.01  0.01                               2039     -1.67   -0.83   0.12  0.59  -1.15 0.02                               ______________________________________                                         NT--Kindred not typed for mfd188.                                        

Using a criterion for linkage to 17q of a LOD score>1.0 for at least onelocus under the CASH model (Claus et al., 1991), four of the 19 kindredsappeared to be linked to 17q (K1901, K1925, K2035, K2082). A number ofadditional kindreds showed some evidence of linkage but at this timecould not be definitively assigned to the linked category. Theseincluded kindreds K1911, K2073, K2039, and K2080. Three of the17q-linked kindreds had informative recombinants in this region andthese are detailed below.

Kindred 2082 is the largest 17q-linked breast cancer family reported todate by any group. The kindred contains 20 cases of breast cancer, andten cases of ovarian cancer. Two cases have both ovarian and breastcancer. The evidence of linkage to 17q for this family is overwhelming;the LOD score with the linked haplotype is over 6.0, despite theexistence of three cases of breast cancer which appear to be sporadic,i.e., these cases share no part of the linked haplotype between Mfd15and 42D6. These three sporadic cases were diagnosed with breast cancerat ages 46, 47, and 54. In smaller kindreds, sporadic cancers of thistype greatly confound the analysis of linkage and the correctidentification of key recombinants. The key recombinant in the 2082kindred is a woman who developed ovarian cancer at age 45 whose motherand aunt had ovarian cancer at ages 58 and 66, respectively. Sheinherited the linked portion of the haplotype for both Mfd188 and 42D6while inheriting unlinked alleles at Mfd15; this recombinant eventplaced BRCA1 distal to Mfd15.

K1901 is typical of early-onset breast cancer kindreds. The kindredcontains 10 cases of breast cancer with a median age at diagnosis of43.5 years of age; four cases were diagnosed under age 40. The LOD scorefor this kindred with the marker 42D6 is 1.5, resulting in a posteriorprobability of 17q-linkage of 0.96. Examination of haplotypes in thiskindred identified a recombinant haplotype in an obligate male carrierand his affected daughter who was diagnosed with breast cancer at age45. Their linked allele for marker Mfd15 differs from that found in allother cases in the kindred (except one case which could not becompletely inferred from her children). The two haplotypes are identicalfor Mfd188 and 42D6. Accordingly, data from Kindred 1901 would alsoplace the BRCA1 locus distal to Mfd15.

Kindred 2035 is similar to K1901 in disease phenotype. The median age ofdiagnosis for the eight cases of breast cancer in this kindred is 37.One case also had ovarian cancer at age 60. The breast cancer cases inthis family descend from two sisters who were both unaffected withbreast cancer until their death in the eighth decade. Each branchcontains four cases of breast cancer with at least one case in eachbranch having markedly early onset. This kindred has a LOD score of 2.34with Mfd5. The haplotypes segregating with breast cancer in the twobranches share an identical allele at Mfd15 but differ for the distalloci Mfd188 and NM23 (a marker typed as part of the consortium which islocated just distal to 42D6 (Hall et al., 1992)). Although the twohaplotypes are concordant for marker 42D6, it is likely that the allelesare shared identical by state (the same allele but derived fromdifferent ancestors), rather than identical by descent (derived from acommon ancestor) since the shared allele is the second most commonallele observed at this locus. By contrast the linked allele shared atMfd15 has a frequency of 0.04. This is a key recombinant in our datasetas it is the sole recombinant in which BRCA1 segregated with theproximal portion of the haplotype, thus setting the distal boundary tothe BRCA1 region. For this event not to be a key recombinant requiresthat a second mutant BRCA1 gene be present in a spouse marrying into thekindred who also shares the rare Mfd15 allele segregating with breastcancer in both branches of the kindred. This event has a probability ofless than one in a thousand. The evidence from this kindred thereforeplaced the BRCA1 locus proximal to Mfd188.

EXAMPLE 3 Creation of a Fine Structure Map and Refinement of the BRCA1Region to Mfd191-Mfd188 using Additional STR Polymorphisms

In order to improve the characterization of our recombinants and definecloser flanking markers, a dense map of this relatively small region onchromosome 17q was required. The chromosome 17 workshop has produced aconsensus map of this region (FIG. 1) based on a combination of geneticand physical mapping studies (Fain, 1992). This map contains both highlypolymorphic STR polymorphisms, and a number of nonpolymorphic expressedgenes. Because this map did not give details on the evidence for thisorder nor give any measure of local support for inversions in the orderof adjacent loci, we viewed it as a rough guide for obtaining resourcesto be used for the development of new markers and construction of ourown detailed genetic and physical map of a small region containingBRCA1. Our approach was to analyze existing STR markers provided byother investigators and any newly developed markers from our laboratorywith respect to both a panel of meiotic (genetic) breakpoints identifiedusing DNA from the CEPH reference families and a panel of somatic cellhybrids (physical breakpoints) constructed for this region. Thesemarkers included 26C2 developed in our laboratory which maps proximal toMfd15, Mfd191 (provided by James Weber), THRA1 (Futreal et al., 1992a),and three polymorphisms kindly provided to us by Dr. Donald Black, NM23(Hall et al. 1992), SCG40 (D17S181), and 6C1 (D17S293).

Genetic localization of markers. In order to localize new markersgenetically within the region of interest, we have identified a numberof key meiotic breakpoints within the region, both in the CEPH referencepanel and in our large breast cancer kindred (K2082). Given the smallgenetic distance in this region, they are likely to be only a relativelysmall set of recombinants which can be used for this purpose, and theyare likely to group markers into sets. The orders of the markers withineach set can only be determined by physical mapping. However the numberof genotypings necessary to position a new marker is minimized. Thesebreakpoints are illustrated in Tables 3 and 4. Using this approach wewere able to genetically order the markers THRA1, 6C1, SCG40, andMfd191. As can be seen from Tables 3 and 4, THRA1 and MFD191 both mapinside the Mfd15-Mfd188 region we had previously identified ascontaining the BRCA1 locus. In Tables 3 and 4, M/P indicates a maternalor paternal recombinant. A "1" indicates inherited allele is ofgrandpaternal origin, while a "0" indicates grandmatemal origin, and "-"indicates that the locus was untyped or uninformative.

                                      TABLE 3                                     __________________________________________________________________________    CEPH Recombinants                                                             Family                                                                            ID  M/P                                                                              Mfd15                                                                             THRA1                                                                             Mfd191                                                                            Mfd188                                                                            SCG40                                                                             6C1                                                                              42D6                                        __________________________________________________________________________    13292                                                                             4   M  1   1   1   0   0   0  0                                           13294                                                                             4   M  1   1   1   0   0   0  0                                           13294                                                                             6   M  0   0   1   1   --  -- --                                          1334                                                                              3   M  1   1   1   1   1   0  0                                           1333                                                                              4   M  1   1   1   0   --  -- 0                                           1333                                                                              6   M  0   0   1   1   --  -- 1                                           1333                                                                              8   P  1   0   0   0   --  -- 0                                           1377                                                                              8   M  0   --  0   0   0   0  1                                           __________________________________________________________________________

                  TABLE 4                                                         ______________________________________                                        Kindred 2082 Recombinants                                                     Family                                                                              ID    M/P    Mfd15 Mfd191                                                                              Mfd188                                                                              SCG40 6C1  42D6                          ______________________________________                                        75          M      0     1     1     1     --   --                            63          M      0     0     1     1     --   1                             125         M      1     1     1     0     --   0                             40          M      1     1     0     0     --   0                             ______________________________________                                    

Analysis of markers Mfd15, Mfd188, Mfd191, and THRA1 in our recombinantfamilies. Mfd15, Mfd188, Mfd191 and THRA1 were typed in our recombinantfamilies and examined for additional information to localize the BRCA1locus. In kindred 1901, the Mfd15 recombinant was recombinant for THRA1but uninformative for Mfd191, thus placing BRCA1 distal to THRA1. InK2082, the recombinant with Mfd15 also was recombinant with Mfd191, thusplacing the BRCA1 locus distal to Mfd191 (Goldgar et al., 1994).Examination of THRA1 and Mfd191 in kindred K2035 yielded no furtherlocalization information as the two branches were concordant for bothmarkers. However, SCG40 and 6C1 both displayed the same pattern asMfd188, thus increasing our confidence in the localization informationprovided by the Mfd188 recombinant in this family. The BRCA1 locus, orat least a portion of it, therefore lies within an interval bounded byMfd191 on the proximal side and Mfd188 on the distal side.

EXAMPLE 4 Development of Genetic and Physical Resources in the Region ofInterest

To increase the number of highly polymorphic loci in the Mfd191-Mfd188region, we developed a number of STR markers in our laboratory fromcosmids and YACs which physically map to the region. These markersallowed us to further refine the region.

STSs were identified from genes known to be in the desired region toidentify YACs which contained these loci, which were then used toidentify subclones in cosmids, P1s or BACs. These subclones were thenscreened for the presence of a CA tandem repeat using a (CA)_(n)oligonucleotide (Pharmacia). Clones with a strong signal were selectedpreferentially, since they were more likely to represent CA-repeatswhich have a large number of repeats and/or are of near-perfect fidelityto the (CA)_(n) pattern. Both of these characteristics are known toincrease the probability of polymorphism (Weber, 1990). These cloneswere sequenced directly from the vector to locate the repeat. Weobtained a unique sequence on one side of the CA-repeat by using one ofa set of possible primers complementary to the end of a CA-repeat, suchas (GT)₁₀ T. Based on this unique sequence, a primer was made tosequence back across the repeat in the other direction, yielding aunique sequence for design of a second primer flanking the CA-repeat.STRs were then screened for polymorphism on a small group of umelatedindividuals and tested against the hybrid panel to confirm theirphysical localization. New markers which satisfied these criteria werethen typed in a set of 40 unrelated individuals from the Utah and CEPHfamilies to obtain allele frequencies appropriate for the studypopulation. Many of the other markers reported in this study were testedin a smaller group of CEPH unrelated individuals to obtain similarlyappropriate allele frequencies.

Using the procedure described above, a total of eight polymorphic STRswas found from these YACS. Of the loci identified in this manner, fourwere both polymorphic and localized to the BRCA1 region. Four markersdid not localize to cliromosome 17, reflecting the chimeric nature ofthe YACs used. The four markers which were in the region were denotedAA1, ED2, 4-7, and YM29. AA1 and ED2 were developed from YACs positivefor the RNU2 gene, 4-7 from an EPB3 YAC and YM29 from a cosmid whichlocalized to the region by the hybrid panel. A description of the numberof alleles, heterozygosity and source of these four and all other STRpolymorphisms analyzed in the breast cancer kindreds is given below inTable 5.

                  TABLE 5                                                         ______________________________________                                        Polymorphic Short Tandem Repeat Markers Used                                  for Fine Structure Mapping of the BRCA1 Locus                                                    Allele* Frequency (%)                                      Clone Gene     Na**   Heterozygosity                                                                         1   2   3   4   5   6                          ______________________________________                                        Mfd15 D17S250  10     0.82     26  22  15   7  7   23                         THRA1 THRA1     5                                                             Mfd191                                                                              D17S776   7     0.55     48  20  11   7  7    7                         ED2   D17S1327 12     0.55     62   9   8   5  5   11                         AA1   D17S1326  7     0.83     28  28  25   8  6    5                         CA375 D17S184  10     0.75     26  15  11   9  9   20                         4-7   D17S1183  9     0.50     63  15   8   6  4    4                         YM29  --        9     0.62     42  24  12   7  7    8                         Mfd188                                                                              D17S579  12     0.92     33  18   8   8  8   25                         SCG40 D17S181  14     0.90     20  18  18  10  8   35                         42D6  D17S588  11     0.86     21  17  11  10  9   32                         6C1   D17S293   7     0.75     30  30  11  11  9    9                         Z109  D17S750   9     0.70     33  27   7   7  7   19                         tdj1475                                                                             D17S1321 13     0.84     21  16  11  11  8   33                         CF4   D17S1320  6     0.63     50  27   9   7  4    3                         tdj1239                                                                             D17S1328 10     0.80     86  10   9   7  4   14                         U5    D17S1325 13     0.83     19  16  12  10  9   34                         ______________________________________                                         *Allele codes 1-5 are listed in decreasing frequency; allele numbers do       not correspond to fragment sizes. Allele 6 frequency is the joint             frequency of all other alleles for each locus.                                **Number of alleles seen in the genetically independent DNA samples used      for calculating allele frequencies.                                      

The four STR polymorphisms which mapped physically to the region (4-7,ED2, AA1, YM29) were analyzed in the meiotic, breakpoint panel showninitially in Tables 3 and 4. Tables 6 and 7 contain the relevant CEPHdata and Kindred 2082 data for localization of these four markers. Inthe tables, M/P indicates a maternal or paternal recombinant. A "1"indicates inherited allele is of grandpaternal origin, while a "0"indicates grandmaternal origin, and "-" indicates that the locus wasuntyped or uninformative.

                                      TABLE 6                                     __________________________________________________________________________    Key Recombinants Used for Genetic Ordering of New STR Loci                    Developed in Our Laboratory Within the BRCA1 Region of 17q                    CEPH                                                                          Family                                                                            ID                                                                              M/P                                                                              Mfd15                                                                             THRA1                                                                             MFD191                                                                             ED2                                                                              AA1                                                                              Z109                                                                             4-7                                                                              YM29                                                                              Mfd188                                                                             SCG40                                                                             42D6                           __________________________________________________________________________    13292                                                                             4 M  1   1   1    1  1  0  0  0   0    0   0                              13294                                                                             4 M  1   0   0    -- 0  -- -- --  0    --  --                             13294                                                                             6 M  0   0   1    -- 1  -- -- --  1    --  --                             1333                                                                              4 M  1   1   1    -- 0  -- -- 0   0    --  0                              1333                                                                              6 M  0   0   1    -- 1  -- -- 1   1    --  1                              1333                                                                              3 M  0   0   1    -- -- -- 1  1   1    --  1                              __________________________________________________________________________

                                      TABLE 7                                     __________________________________________________________________________    Kindred 2082 Recombinants                                                     ID M/P                                                                              Mfd15                                                                             Mfd191                                                                            ED2                                                                              AA1 4-7                                                                              YM29                                                                              Mfd188                                                                            SCG40                                                                             42D6                                      __________________________________________________________________________    63 M  0   0   1  --  1  1   1   1   1                                         125                                                                              M  1   1   1  --  1  1   1   0   0                                         40 M  1   1   0  --  0  --  0   0   0                                         22 P  0   0   1  1   1  1   1   1   1                                         __________________________________________________________________________

From CEPH 1333-04, we see that AA1 and YM29 must lie distal to Mfd191.From 13292, it can be inferred that both AA1 and ED2 are proximal to4-7, YM29, and Mfd188. The recombinants found in K2082 provide someadditional ordering information. Three independent observations(individual numbers 22, 40, & 63) place AA1, ED2, 4-7, and YM29, andMfd188 distal to Mfd191, while ID 125 places 4-7, YM29, and Mfd188proximal to SCG40. No genetic information on the relative orderingwithin the two clusters of markers AA1/ED2 and 4-7/YM29/Mfd188 wasobtained from the genetic recombinant analysis. Although ordering lociwith respect to hybrids which are known to contain "holes" in whichsmall pieces of interstitial human DNA may be missing is problematic,the hybrid patterns indicate that 4-7 lies above both YM29 and Mfd188.

EXAMPLE 5 Genetic Analyses of Breast Cancer Kindreds with MarkersAA1,4-7, ED2, and YM29

In addition to the three kindreds containing key recombinants which havebeen discussed previously, kindred K2039 was shown through analysis ofthe newly developed STR markers to be linked to the region and tocontain a useful recombinant.

Table 8 defines the haplotypes (shown in coded form) of the kindreds interms of specific marker alleles at each locus and their respectivefrequencies. In Table 8, alleles are listed in descending order offrequency; frequencies of alleles 1-5 for each locus are given in Table5. Haplotypes coded H are BRCA1 associated haplotypes, P designates apartial H haplotype, and an R indicates an observable recombinanthaplotype. As evident in Table 8, not all kindreds were typed for allmarkers; moreover, not all individuals within a kindred were typed foran identical set of markers, especially in K2082. With one exception,only haplotypes inherited from affected or at-risk kindred members areshown; haplotypes from spouses marrying into the kindred are notdescribed. Thus in a given sibship, the appearance of haplotypes X and Yindicates that both haplotypes from the affected/at-risk individual wereseen and neither was a breast cancer associated haplotype.

                                      TABLE 8                                     __________________________________________________________________________    Breast Cancer Linked Haplotypes Found in the Three Kindreds                   Kin.                                                                             HAP                                                                              Mfd15                                                                             THRA1                                                                             Mfd191                                                                            tdj1475                                                                           ED2                                                                              AA1                                                                              Z109                                                                             CA375                                                                             4-7                                                                              YM29                                                                              Mfd188                                                                            SCG40                                                                             6C1                                                                              42D6                     __________________________________________________________________________    1901                                                                             H1 1   5   5   3   1  4  NI NI  1  1   3   NI  NI 1                           R2 9   2   5   6   1  4  NI NI  1  1   3   NI  NI 1                        2082                                                                             H1 3   NI  4   6   6  1  NI NI  2  1   4   2   NI 1                           P1 3   NI  4   NI  NI NI NI NI  NI NI  4   2   NI 1                           P2 3   NI  NI  NI  NI NI NI NI  NI NI  4   NI  NI NI                          R1 6   NI  1   5   6  1  NI NI  2  1   4   2   NI 1                           R2 6   NI  4   6   6  1  NI NI  2  1   4   2   NI 1                           R3 3   NI  4   NI  6  1  NI NI  2  1   4   1   NI 7                           R4 7   NI  1   NI  1  5  NI NI  4  6   1   2   NI 1                           R5 3   NI  4   NI  NI NI NI NI  NI 2   1   NI  NI NI                          R6 3   NI  4   3   1  2  NI NI  1  2   2   6   NI 6                           R7 3   NI  4   3   7  1  NI NI  1  1   3   7   NI 4                        2035                                                                             HI 8   2   1   NI  5  1  1  4   3  1   6   8   2  4                           H2 8   2   1   NI  5  1  1  2   1  1   2   3   1  4                           R2 8   2   1   NI  5  1  1  2   1  1   2   3   6  1                        __________________________________________________________________________

In kindred K1901, the new markers showed no observable recombinationwith breast cancer susceptibility, indicating that the recombinationevent in this kindred most likely took place between THRA1 and ED2.Thus, no new BRCA1 localization information was obtained based uponstudying the four new markers in this kindred. In kindred 2082 the keyrecombinant individual has inherited the linked alleles for ED2, 4-7,AA1, and YM29, and was recombinant for tdj1474 indicating that therecombination event occurred in this individual between tdj1474 andED2/AA1.

There are three haplotypes of interest in kindred K2035, H1, H2, and R2shown in Table 8. H1 is present in the four cases and one obligate malecarrier descendant from individual 17 while H2 is present or inferred intwo cases and two obligate male carriers in descendants of individual10. R2 is identical to H2 for loci between and including Mfd15 andSCG40, but has recombined between SCG40 and 42D6. Since we haveestablished that BRCA1 is proximal to 42D6, this H2/R2 difference addsno further localization information. H1 and R2 share an identical alleleat Mfd15, THRA1, AA1, and ED2 but differ for loci presumed distal toED2, i.e., 4-7, Mfd188, SCG40, and 6C1. Although the two haplotypes areconcordant for the 5th allele for marker YM29, a marker which mapsphysically between 4-7 and Mfd 188, it is likely that the alleles areshared identical by state rather than identical by descent since thisallele is the most common allele at this locus with a frequencyestimated in CEPH parents of 0.42. By contrast, the linked allelesshared at the Mfd15 and ED2 loci have frequencies of 0.04 and 0.09,respectively. They also share more common alleles at Mfd191(frequency=0.52), THRA1, and AA1 (frequency=0.28). This is the keyrecombinant in the set as it is the sole recombinant in which breastcancer segregated with the proximal portion of the haplotype, thussetting the distal boundary. The evidence from this kindred thereforeplaces the BRCA1 locus proximal to 4-7.

The recombination event in kindred 2082 which places BRCA1 distal totdj1474 is the only one of the four events described which can bedirectly inferred; that is, the affected mother's genotype can beinferred from her spouse and offspring, and the recombinant haplotypecan be seen in her affected daughter. In this family the odds in favorof affected individuals carrying BRCA1 susceptibility alleles areextremely high; the only possible interpretations of the data are thatBRCA1 is distal to Mfd191 or alternatively that the purportedrecombinant is a sporadic case of ovarian cancer at age 44. Rather thana directly observable or inferred recombinant, interpretation of kindred2035 depends on the observation of distinct 17q-haplotypes segregatingin different and sometimes distantly related branches of the kindred.The observation that portions of these haplotypes have alleles in commonfor some markers while they differ at other markers places the BRCA1locus in the shared region. The confidence in this placement depends onseveral factors: the relationship between the individuals carrying therespective haplotypes, the frequency of the shared allele, the certaintywith which the haplotypes can be shown to segregate with the BRCA1locus, and the density of the markers in the region which define thehaplotype. In the case of kindred 2035, the two branches are closelyrelated, and each branch has a number of early onset cases which carrythe respective haplotype. While two of the shared alleles are common,(Mfd191, THRA1), the estimated frequencies of the shared alleles atMfd15, AA1, and ED2 are 0.04, 0.28, and 0.09, respectively. It istherefore highly likely that these alleles are identical by descent(derived from a common ancestor) rather than identical by state (thesame allele but derived from the general population).

EXAMPLE 6 Refined Physical Mapping Studies Place the BRCA1 Gene in aRegion Flanked by tdj1474 and U5R

Since its initial localization to chromosome 17q in 1990 (Hall et al.,1990) a great deal of effort has gone into localizing the BRCA1 gene toa region small enough to allow implementation of effective positionalcloning strategies to isolate the gene. The BRCA1 locus was firstlocalized to the interval Mfd15 (D17S250)-42D6 (D17S588) by multipointlinkage analysis (Easton et al., 1993) in the collaborative BreastCancer Linkage Consortium dataset consisting of 214 families collectedworldwide. Subsequent refinements of the localization have been basedupon individual recombinant events in specific families. The regionTHRA1-D17S183 was defined by Bowcock et al., 1993; and the regionTHRA1-D17S78 was defined by Simard et al., 1993.

We further showed that the BRCA1 locus must lie distal to the markerMfd191 (D17S776) (Goldgar et al., 1994). This marker is known to liedistal to THRA1 and RARA. The smallest published region for the BRCA1locus is thus between D17S776 and D17S78. This region still containsapproximately 1.5 million bases of DNA, making the isolation and testingof all genes in the region a very difficult task. We have thereforeundertaken the tasks of constructing a physical map of the region,isolating a set of polymorphic STR markers located in the region, andanalyzing these new markers in a set of informative families to refinethe location of the BRCA1 gene to a manageable interval.

Four families provide important genetic evidence for localization ofBRCA1 to a sufficiently small region for the application of positionalcloning strategies. Two families (K2082, K1901) provide data relating tothe proximal boundary for BRCA1 and the other two (K2035, K1813) fix thedistal boundary. These families are discussed in detail below. A totalof 15 Short Tandem Repeat markers assayable by PCR were used to refinethis localization in the families studied. These markers includeDS17S7654, DS17S975, tdj1474, and tdj1239. Primer sequences for thesemarkers are provided in SEQ ID NO:3 and SEQ ID NO:4 for DS17S754; in SEQID NO:5 and SEQ ID NO:6 for DS17S975; in SEQ ID NO:7 and SEQ ID NO:8 fortdj1474; and, in SEQ ID NO:9 and SEQ ID NO:10 for tdj1239.

Kindred 2082

Kindred 2082 is the largest BRCA1-linked breast/ovarian cancer familystudied to date. It has a LOD score of 8.6, providing unequivocalevidence for 17q linkage. This family has been previously described andshown to contain a critical recombinant placing BRCA1 distal to MFD191(D17S776). This recombinant occurred in a woman diagnosed with ovariancancer at age 45 whose mother had ovarian cancer at age 63. The affectedmother was deceased; however, from her children, she could be inferredto have the linked haplotype present in the 30 other linked cases in thefamily in the region between Mfd15 and Mfd188. Her affected daughterreceived the linked allele at the loci ED2, 4-7, and Mfd188, butreceived the allele on the non-BRCA1 chromosome at Mfd15 and Mfd91. Inorder to further localize this recombination breakpoint, we tested DNAfrom the key members of this family for the following markers derivedfrom physical mapping resources: tdj1474, tdj1239, CF4, D17S855. For themarkers tdj1474 and CF4, the affected daughter did not receive thelinked allele. For the STR locus tdj1239, however, the mother could beinferred to be informative and her daughter did receive theBRCA1-associated allele. D17S855 was not informative in this family.Based on this analysis, the order is 17qcentromere-Mfd191-17HSD-CF4-tdj1474-tdj1239-D17S855-ED2-4-7-Mfd188-17qtelomere. The recombinant described above therefore places BRCA1 distalto tdj1474, and the breakpoint is localized to the interval betweentdj1474 and tdj1239. The only alternative explanation for the data inthis family other than that of BRCA1 being located distal to tdj1474, isthat the ovarian cancer present in the recombinant individual is causedby reasons independent of the BRCA1 gene. Given that ovarian cancerdiagnosed before age 50 is rare, this alternate explanation isexceedingly unlikely.

Kindred 1901

Kindred 1901 is an early-onset breast cancer family with 7 cases ofbreast cancer diagnosed before 50, 4 of which were diagnosed before age40. In addition, there were three cases of breast cancer diagnosedbetween the ages of 50 and 70. One case of breast cancer also hadovarian cancer at age 61. This family currently has a LOD score of 1.5with D17S855. Given this linkage evidence and the presence of at leaseone ovarian cancer case, this family has a posterior probability ofbeing due to BRCA1 of over 0.99. In this family, the recombination comesfrom the fact that an individual who is the brother of the ovariancancer case from which the majority of the other cases descend, onlyshares a portion of the haplotype which is cosegregating with the othercases in the family. However, he passed this partial haplotype to hisdaughter who developed breast cancer at age 44. If this case is due tothe BRCA1 gene, then only the part of the haplotype shared between thisbrother and his sister can contain the BRCA1 gene. The difficulty ininterpretation of this kind of information is that while one can be sureof the markers which are not shared and therefore recombinant, markerswhich are concordant can either be shared because they arenon-recombinant, or because their parent was homozygous. Without theparental genotypic data it is impossible to discriminate between thesealternatives. Inspection of the haplotype in K1901, shows that he doesnot share the linked allele at Mfd15 (D17S250), THRA1, CF4 (D17S1320),and tdj1474 (17DS1321). He does share the linked allele at Mfd191(D17S776), ED2 (D17S1327), tdj1239 (D17S1328), and Mfd188 (D17S579).Although the allele shared at Mfd191 is relatively rare (0.07), we wouldpresume that the parent was homozygous since they are recombinant withmarkers located nearby on either side, and a double recombination eventin this region would be extremely unlikely. Thus the evidence in thisfamily would also place the BRCA1 locus distal to tdj474. However, thelower limit of this breakpoint is impossible to determine withoutparental genotype information. It is intriguing that the key recombinantbreakpoint in this family confirms the result in Kindred 2082. Asbefore, the localization information in this family is only meaningfulif the breast cancer was due to the BRCA1 gene. However, her relativelyearly age at diagnosis (44) makes this seem very likely since the riskof breast cancer before age 45 in the general population is low(approximately 1%).

Kindred 2035

This family is similar to K1901 in that the information on the criticalrecombinant events is not directly observed but is inferred from theobservation that the two haplotypes which are cosegregating with theearly onset breast cancer in the two branches of the family appearidentical for markers located in the proximal portion of the 17q BRCA1region but differ at more distal loci. Each of these two haplotypesoccurs in at least four cases of early-onset or bilateral breast cancer.The overall LOD score with ED2 in this family is 2.2, and consideringthat there is a case of ovarian cancer in the family (indicating a priorprobability of BRCA1 linkage of 80%), the resulting posteriorprobability that this family is linked to BRCA1 is 0.998. The haplotypesare identical for the markers Mfd15, THRA1, Mfd191, ED2, AA1, D17S858and D17S902. The common allele at Mfd15 and ED2 are both quite rare,indicating that this haplotype is shared identical by descent. Thehaplotypes are discordant, however, for CA375, 4-7, and Mfd188, andseveral more distal markers. This indicates that the BRCA1 locus mustlie above the marker CA-375. This marker is located approximately 50 kbbelow D17S78, so it serves primarily as additional confirmation of thisprevious lower boundary as reported in Simard et al. (1993).

Kindred 1813

Kindred 1813 is a small family with four cases of breast cancerdiagnosed at very early ages whose mother also had breast cancerdiagnosed at an early age and ovarian cancer some years later. Thisfamily yields a maximum multipoint LOD score of 0.60 with 17q markersand, given that there is at least one case of ovarian cancer, results ina posterior probability of being a BRCA1 linked family of 0.93. Thisfamily contains a directly observable recombination event in individual18 (see FIG. 5 in Simard et al., Human Mol. Genet. 2:1193-1199 (1993)),who developed breast cancer at age 34. The genotype of her affectedmother at the relevant 17q loci can be inferred from her genotypes, heraffected sister's genotypes, and the genotypes of three other unaffectedsiblings. Individual 18 inherits the BRCA1-linked alleles for thefollowing loci: Mfd15, THRA1, D17S800, D17S855, AA1, and D17S931.However, for markers below D17S931, i.e., U5R, vrs31, D17S858, andD17S579, she has inherited the alleles located on the non-diseasebearing chromosome. The evidence from this family therefore would placethe BRCA1 locus proximal to the marker U5R. Because of her early age atdiagnosis (34) it is extremely unlikely that the recombinantindividual's cancer is not due to the gene responsible for the othercases of breast/ovarian cancer in this family; the uncertainty in thisfamily comes from our somewhat smaller amount of evidence that breastcancer in this family is due to BRCA1 rather than a second, as yetunmapped, breast cancer susceptibility locus.

Size of the region containing BRCA1

Based on the genetic data described in detail above, the BRCA1 locusmust lie in the interval between the markers tdj1474 and U5R, both ofwhich were isolated in our laboratory. Based upon the physical mapsshown in FIGS. 2 and 3, we can try to estimate the physical distancebetween these two loci. It takes approximately 14 P1 clones with anaverage insert size of approximately 80 kb to span the region. However,because all of these P1s overlap to some unknown degree, the physicalregion is most likely much smaller than 14 times 80 kb. Based onrestriction maps of the clones covering the region, we estimate the sizeof the region containing BRCA1 to be approximately 650 kb.

EXAMPLE 7 Identification of Candidate cDNA Clones for the BRCA1 Locus byGenomic Analysis of the Contig Region

Complete screen of the plausible region. The first method to identifycandidate cDNAs, although labor intensive, used known techniques. Themethod comprised the screening of cosmids and P1 and BAC clones in thecontig to identify putative coding sequences. The clones containingputative coding sequences were then used as probes on filters of cDNAlibraries to identify candidate cDNA clones for future analysis. Theclones were screened for putative coding sequences by either of twomethods.

Zoo blots. The first method for identifying putative coding sequenceswas by screening the cosmid and P1 clones for sequences conservedthrough evolution across several species. This technique is referred toas "zoo blot analysis" and is described by Monaco, 1986. Specifically,DNAs from cow, chicken, pig, mouse and rat were digested with therestriction enzymes EcoRi and HindlIl (8 μg of DNA per enzyme). Thedigested DNAs were separated overnight on an 0.7% gel at 20 volts for 16hours (14 cm gel), and the DNA transferred to Nylon membranes usingstandard Southern blot techniques. For example, the zoo blot filter wastreated at 65° C. in 0.1× SSC, 0.5% SDS, and 0.2M Tris, pH 8.0, for 30minutes and then blocked overnight at 42° C. in 5× SSC, 10% PEG 8000, 20mM NaPO₄ pH 6.8, 100 μg/ml Salmon Sperm DNA, 1x Denhardt's, 50%formamide, 0.1% SDS, and 2 μg/ml C_(o) t-1 DNA.

The cosmid and P1 clones to be analyzed were digested with a restrictionenzyme to release the human DNA from the vector DNA. The DNA wasseparated on a 14 cm, 0.5% agarose gel run overnight at 20 volts for 16hours. The human DNA bands were cut out of the gel and electroelutedfrom the gel wedge at 100 volts for at least two hours in 0.5x TrisAcetate buffer (Maniatis et al., 1982). The eluted Not I digested DNA(˜15 kb to 25 kb) was then digested with EcoRI restriction enzyme togive smaller fragments (˜0.5 kb to 5.0 kb) which melt apart more easilyfor the next step of labeling the DNA with radionucleotides. The DNAfragments were labeled by means of the hexamer random prime labelingmethod (Boehringer-Mannheim, Cat. #1004760). The labeled DNA wasspermine precipitated (add 100 μl TE, 5 μl 0.1M spermine, and 5 μl of 10mg/ml salmon sperm DNA) to remove unincorporated radionucleotides. Thelabeled DNA was then resuspended in 100 μl TE, 0.5M NaCI at 65° C. for 5minutes and then blocked with Human C_(o) t-1 DNA for 2-4 hrs. as perthe manufacturer's instructions (Gibco/BRL, Cat. #5279SA). The C_(o) t-1blocked probe was incubated on the zoo blot filters in the blockingsolution overnight at 42° C. The filters were washed for 30 minutes atroom temperature in 2× SSC, 0.1% SDS, and then in the same buffer for 30minutes at 55° C. The filters were then exposed 1 to 3 days at -70° C.to Kodak XAR-5 film with an intensifying screen. Thus, the zoo blotswere hybridized with either the pool of Eco-R1 fragments from theinsert, or each of the fragments individually.

HTF island analvsis. The second method for identifying cosmids to use asprobes on the cDNA libraries was HTF island analysis. Since thepulsed-field map can reveal HTF islands, cosmids that map to these HTFisland regions were analyzed with priority. HTF islands are segments ofDNA which contain a very high frequency of unmethylated CpGdinucleotides (Tonolio et al., 1990) and are revealed by the clusteringof restriction sites of enzymes whose recognition sequences include CpGdinucleotides. Enzymes known to be useful in HTF-island analysis areAscI, NotI, BssHII, EagI, SacII, NaeI, NarI, SmaI, and Mlul (Anand,1992). A pulsed-field map was created using the enzymes NotI, NruI,EagI, SacII, and SaII, and two HTF islands were found. These islands arelocated in the distal end of the region, one being distal to the GP2Blocus, and the other being proximal to the same locus, both outside theBRCA1 region. The cosmids derived from the YACs that cover these twolocations were analyzed to identify those that contain these restrictionsites, and thus the HTF islands.

cDNA screening. Those clones that contain HTF islands or showhybridization to other species DNA besides human are likely to containcoding sequences. The human DNA from these clones was isolated as wholeinsert or as EcoR1 fragments and labeled as described above. The labeledDNA was used to screen filters of various cDNA libraries under the sameconditions as the zoo blots except that the cDNA filters undergo a morestringent wash of 0.1× SSC, 0.1% SDS at 65° C. for 30 minutes twice.

Most of the cDNA libraries used to date in our studies (libraries fromnormal breast tissue, breast tissue from a woman in her eighth month ofpregnancy and a breast malignancy) were prepared at Clonetech, Inc. ThecDNA library generated from breast tissue of an 8 month pregnant womanis available from Clonetech (Cat. #HL1037a) in the Lambda gt-10 vector,and is grown in C600Hfl bacterial host cells. Normal breast tissue andmalignant breast tissue samples were isolated from a 37 year oldCaucasian female and one-gram of each tissue was sent to Clonetech formRNA processing and cDNA library construction. The latter two librarieswere generated using both random and oligo-dT priming, with sizeselection of the final products which were then cloned into the LambdaZap II vector, and grown in XL1-blue strain of bacteria as described bythe manufacturer. Additional tissue-specific cDNA libraries includehuman fetal brain (Stratagene, Cat. 936206), human testis (ClonetechCat. HL3024), human thymus (Clonetech Cat. HL 1127n), human brain(Clonetech Cat. HL11810), human placenta (Clonetech Cat 1075b), andhuman skeletal muscle (Clonetech Cat. HL1124b).

The cDNA libraries were plated with their host cells on NZCYM plates,and filter lifts are made in duplicate from each plate as per Maniatiset al. (1982). Insert (human) DNA from the candidate genomic clones waspurified and radioactively labeled to high specific activity. Theradioactive DNA was then hybridized to the cDNA filters to identifythose cDNAs which correspond to genes located within the candidatecosmid clone. cDNAs identified by this method were picked, replated, andscreened again with the labeled clone insert or its derived EcoR1fragment DNA to verify their positive status. Clones that were positiveafter this second round of screening were then grown up and their DNApurified for Southern blot analysis and sequencing. Clones were eitherpurified as plasmid through in vivo excision of the plasmid from theLambda vector as described in the protocols from the manufacturers, orisolated from the Lambda vector as a restriction fragment and subclonedinto plasmid vector.

The Southern blot analysis was performed in duplicate, one using theoriginal genomic insert DNA as a probe to verify that cDNA insertcontains hybridizing sequences. The second blot was hybridized with cDNAinsert DNA from the largest cDNA clone to identify which clonesrepresent the same gene. All cDNAs which hybridize with the genomicclone and are unique were sequenced and the DNA analyzed to determine ifthe sequences represent known or unique genes. All cDNA clones whichappear to be unique were further analyzed as candidate BRCA1 loci.Specifically, the clones are hybridized to Northern blots to look forbreast specific expression and differential expression in normal versusbreast tumor RNAs. They are also analyzed by PCR on clones in the BRCA1region to verify their location. To map the extent of the locus, fulllength cDNAs are isolated and their sequences used as PCR probes on theYACs and the clones surrounding and including the original identifyingclones. Intron-exon boundaries are then further defined through sequenceanalysis.

We have screened the normal breast, 8 month pregnant breast and fetalbrain cDNA libraries with zoo blot-positive Eco R1 fragments from cosmidBAC and P1 clones in the region. Potential BRCA1 cDNA clones wereidentified among the three libraries. Clones were picked, replated, andscreened again with the original probe to verify that they werepositive.

Analvsis of hybrid-selected cDNA. cDNA fragments obtained from directselection were checked by Southern blot hybridization against the probeDNA to verify that they originated from the contig. Those that passedthis test were sequenced in their entirety. The set of DNA sequencesobtained in this way were then checked against each other to findindependent clones that overlapped. For example, the clones 694-65,1240-1 and 1240-33 were obtained independently and subsequently shown toderive from the same contiguous cDNA sequence which has been namedEST:489:1.

Analvsis of candidate clones. One or more of the candidate genesgenerated from above were sequenced and the information used foridentification and classification of each expressed gene. The DNAsequences were compared to known genes by nucleotide sequencecomparisons and by translation in all frames followed by a comparisonwith known amino acid sequences. This was accomplished using GeneticData Environment (GDE) version 2.2 software and the Basic LocalAlignment Search Tool (Blast) series of client/server software packages(e.g., BLASTN 1.3.1 3MP), for sequence comparison against both local andremote sequence databases (e.g., GenBank), running on Sun SPARCworkstations. Sequences reconstructed from collections of cDNA clonesidentified with the cosmids and P1 s have been generated. All candidategenes that represented new sequences were analyzed further to test theircandidacy for the putative BRCA1 locus.

Mutation screening. To screen for mutations in the affected pedigrees,two different approaches were followed. First, genomic DNA isolated fromfamily members known to carry the susceptibility allele of BRCA1 wasused as a template for amplification of candidate gene sequences by PCR.If the PCR primers flank or overlap an intron/exon boundary, theamplified fragment will be larger than predicted from the cDNA sequenceor will not be present in the amplified mixture. By a combination ofsuch amplification experiments and sequencing of P1, BAC or cosmidclones using the set of designed primers it is possible to establish theintron/exon structure and ultimately obtain the DNA sequences of genomicDNA from the pedigrees.

A second approach that is much more rapid if the intron/exon structureof the candidate gene is complex involves sequencing fragments amplifiedfrom pedigree lymphocyte cDNA. CDNA synthesized from lymphocyte mRNAextracted from pedigree blood was used as a substrate for PCRamplification using the set of designed primers. If the candidate geneis expressed to a significant extent in lymphocytes, such experimentsusually produce amplified fragments that can be sequenced directlywithout knowledge of intron/exon junctions.

The products of such sequencing reactions were analyzed by gelelectrophoresis to determine positions in the sequence that containeither mutations such as deletions or insertions, or base pairsubstitutions that cause amino acid changes or other detrimentaleffects.

Any sequence within the BRCA1 region that is expressed in breast isconsidered to be a candidate gene for BRCA1. Compelling evidence that agiven candidate gene corresponds to BRCA1 comes from a demonstrationthat pedigree families contain defective alleles of the candidate.

EXAMPLE 8 Identification of BRCA1

Identification of BRCA1. Using several strategies, a detailed map oftranscripts was developed for the 600 kb region of 17q21 betweenD17S1321 and D17S1324. Candidate expressed sequences were defined as DNAsequences obtained from: 1) direct screening of breast, fetal brain, orlymphocyte cDNA libraries, 2) hybrid selection of breast, lymphocyte orovary cDNAs, or 3) random sequencing of genomic DNA and prediction ofcoding exons by XPOUND (Thomas and Skolnick, 1994). These expressedsequences in many cases were assembled into contigs composed of severalindependently identified sequences. Candidate genes may comprise morethan one of these candidate expressed sequences. Sixty-five candidateexpressed sequences within this region were identified by hybridselection, by direct screening of cDNA libraries, and by randomsequencing of P1 subclones. Expressed sequences were characterized bytranscript size, DNA sequence, database comparison, expression pattern,genomic structure, and, most importantly, DNA sequence analysis inindividuals from kindreds segregating 17q-linked breast and ovariancancer susceptibility.

Three independent contigs of expressed sequence, 1141:1 (649 bp), 694:5(213 bp) and 754:2 (1079 bp) were isolated and eventually shown torepresent portions of BRCA1. When ESTs for these contigs were used ashybridization probes for Northern analysis, a single transcript ofapproximately 7.8 kb was observed in normal breast mRNA, suggesting thatthey encode different portions of a single gene. Screens of breast,fetal brain, thymus, testes, lymphocyte and placental cDNA libraries andPCR experiments with breast mRNA linked the 1141:1, 694:5 and 754:2contigs. 5' RACE experiments with thymus, testes, and breast mRNAextended the contig to the putative 5' end, yielding a composite fulllength sequence. PCR and direct sequencing of P1s and BACs in the regionwere used to identify the location of introns and allowed thedetermination of splice donor and acceptor sites. These three expressedsequences were merged into a single transcription unit that proved inthe final analysis to be BRCA1. This transcription unit is locatedadjacent to D17S855 in the center of the 600 kb region (FIG. 4).

Combination of sequences obtained from cDNA clones, hybrid selectionsequences, and amplified PCR products allowed construction of acomposite full length BRCA1 cDNA (SEQ ID NO:1). The sequence of theBRCA1 cDNA (up through the stop codon) has also been deposited withGenBank and assigned accession number U-14680. This deposited sequenceis incorporated herein by reference. The cDNA clone extending farthestin the 3' direction contains a poly(A) tract preceded by apolyadenylation signal. Conceptual translation of the cDNA revealed asingle long open reading frame of 208 kilodaltons (amino acid sequence:SEQ ID NO:2) with a potential initiation codon flanked by sequencesresembling the Kozak consensus sequence (Kozak, 1987). Smith-Waterman(Smith and Waterman, 1981) and BLAST (Altschul et al., 1990) searchesidentified a sequence near the amino terminus with considerable homologyto zinc-finger domains (FIG. 5). This sequence contains cysteine andhistidine residues present in the consensus C3HC4 zinc-finger motif andshares multiple other residues with zinc-finger proteins in thedatabases. The BRCA1 gene is composed of 23 coding exons arrayed overmore than 100 kb of genomic DNA (FIG. 6). Northern blots using fragmentsof the BRCA1 cDNA as probes identified a single transcript of about 7.8kb, present most abundantly in breast, thymus and testis, and alsopresent in ovary (FIG. 7). Four alternatively spliced products wereobserved as independent cDNA clones; 3 of these were detected in breastand 2 in ovary mRNA (FIG. 6). A PCR survey from tissue cDNAs furthersupports the idea that there is considerable heterogeneity near the 5'end of transcripts from this gene; the molecular basis for theheterogeneity involves differential choice of the first splice donorsite, and the changes detected all alter the transcript in the region 5'of the identified start codon. We have detected six potential alternatesplice donors in this 5' untranslated region, with the longest deletionbeing 1,155 bp. The predominant form of the BRCA1 protein in breast andovary lacks exon 4. The nucleotide sequence for BRCA1 exon 4 is shown inSEQ ID NO:11, with the predicted amino acid sequence shown in SEQ IDNO:12.

Additional 5' sequence of BRCA1 genomic DNA is set forth in SEQ IDNO:13. The G at position 1 represents the potential start site intestis. The A in position 140 represents the potential start site insomatic tissue. There are six alternative splice forms of this 5'sequence as shown in FIG. 8. The G at position 356 represents thecanonical first splice donor site. The G at position 444 represents thefirst splice donor site in two clones (testis 1 and testis 2). The G atposition 889 represents the first splice donor site in thymus 3. Afourth splice donor site is the G at position 1230. The T at position1513 represents the splice acceptor site for all of the above splicedonors. A fifth alternate splice form has a first splice donor site atposition 349 with a first acceptor site at position 591 and a secondsplice donor site at position 889 and a second acceptor site at position1513. A sixth alternate form is unspliced in this 5' region. The A atposition 1532 is the canonical start site, which appears at position 120of SEQ ID NO:1. Partial genomic DNA sequences determined for BRCA1 areset forth in FIGS. 10A-10H and SEQ ID Numbers:14-34. The lower caseletters (in FIGS. 10A-10H) denote intron sequence while the upper caseletters denote exon sequence. Indefinite intervals within introns aredesignated with vvvvvvvvvvvvv in FIGS. 10A-10H. The intron/exonjunctions are shown in Table 9. The CAG found at the 5' end of exons 8and 14 is found in some cDNAs but not in others. Known polymorphic sitesare shown in FIGS. 10A-10H in boldface type and are underlined.

                                      TABLE 9                                     __________________________________________________________________________        Base                                                                      Exon                                                                              Position*    Intron Borders                                               No.                                                                              5'   3' Length                                                                              5'                     3'                                    __________________________________________________________________________    e1   1   100                                                                             100   GATAAATTAAAACTGCGACTGCGCGGCGTG.sup.35*                                                               GTAGTAGAGTCCCGGGAAAGGGACAGGGGG.sup                                            .36                                   e2  101  199                                                                              99   ATATATATATGTTTTTCTAATGTGTTAAAG.sup.37                                                                GTAAGTCAGCACAAGAGTGTATTAATTTGG.sup                                            .38                                   e3  200  253                                                                              54   TTTCTTTTTCTCCCCCCCCTACCCTGCTAG.sup.39                                                                GTAAGTTTGAATGTGTTATGTGGCTCCATT.sup                                            .40                                   e4  ***  ***                                                                             111   AGCTACTTTTTTTTTTTTTTTTTGAGACAG.sup.41                                                                GTAAGTGCACACCACCATATCCAGCTAAAT.sup                                            .42                                   e5  254  331                                                                              78   AATTGTTCTTTCTTTCTTTATAATTTATAG.sup.43                                                                GTATATAATTTGGTAATGATGCTAGGTTGG.sup                                            .44                                   e6  332  420                                                                              89   GAGTGTGTTTCTCAAACAATTTAATTTCAG.sup.45                                                                GTAAGTGTTGAATATCCCAAGAATCACACT.sup                                            .46                                   e7  421  560                                                                             140   AAACATAATGTTTTCCCTTGTATTTTACAG.sup.47                                                                GTAAAACCATTTGTTTTCTTCTTCTTCTTC.sup                                            .48                                   e8  561  666                                                                             106   TGCTTGACTGTTCTTTACCATACTGTTTAG.sup.49                                                                GTAAGGGTCTCAGGTTTTTTAAGTATTTAA.sup                                            .50                                   e9  667  712                                                                              46   TGATTTATTTTTTGGGGGGAAATTTTTTAG.sup.51                                                                GTGAGTCAAAGAGAACCTTTGTCTATGAAG.sup                                            .52                                   e10                                                                               713  789                                                                              77   TCTTATTAGGACTCTGTCTTTTCCCTATAG.sup.53                                                                GTAATGGCAAAGTTTGCCAACTTAACAGGC.sup                                            .54                                   e11                                                                               790 4215                                                                             3426  GAGTACCTTGTTATTTTTGTATATTTTCAG.sup.55                                                                GTATTGGAACCAGGTTTTTGTGTTTGCCCC.sup                                            .56                                   e12                                                                              4216 4302                                                                              87   ACATCTGAACCTCTGTTTTTGTTATTTAAG.sup.57                                                                AGGTAAAAAGCGTGTGTGTGTGTGCACATG.sup                                            .58                                   e13                                                                              4303 4476                                                                             174   CATTTTCTTGGTACCATTTATCGTTTTTGA.sup.59                                                                GTGTGTATTGTTGGCCAAACACTGATATCT.sup                                            .60                                   e14                                                                              4477 4603                                                                             127   AGTAGATTTGTTTTCTCATTCCATTTAAAG.sup.61                                                                GTAAGAAACATCAATGTAAAGATGCTGTGG.sup                                            .62                                   e15                                                                              4604 4794                                                                             191   ATGGTTTTCTCCTTCCATTTATCTTTCTAG.sup.63**                                                              GTAATATTTCATCTGCTGTATTGGAACAAA.sup                                            .64                                   e16                                                                              4795 5105                                                                             311   TGTAAATTAAACTTCTCCCATTCCTTTCAG.sup.65                                                                GTGAGTGTATCCATATGTATCTCCCTAATG.sup                                            .66                                   e17                                                                              5106 5193                                                                              88   ATGATAATGGAATATTTGATTTAATTTCAG.sup.67                                                                GTATACCAAGAACCTTTACAGAATACCTTG.sup                                            .68                                   e18                                                                              5194 5271                                                                              78   CTAATCCTTTGAGTGTTTTTCATTCTGCAG.sup.69                                                                GTAAGTATAATACTATTTCTCCCCTCCTCC.sup                                            .70                                   e19                                                                              5272 5312                                                                              41   TGTAACCTGTCTTTTCTATGATCTCTTTAG.sup.71                                                                GTAAGTACTTGATGTTACAAACTAACCAGA.sup                                            .72                                   e20                                                                              5313 5396                                                                              84   TCCTGATGGGTTGTGTTTGGTTTCTTTCAG.sup.73                                                                GTAAAGCTCCCTCCCTCAAGTTGACAAAAA.sup                                            .74                                   e21                                                                              5397 5451                                                                              55   CTGTCCCTCTCTCTTCCTCTCTTCTTCCAG.sup.75                                                                GTAAGAGCCTGGGAGAACCCCAGAGTTCCA.sup                                            .76                                   e22                                                                              5452 5525                                                                              74   AGTGATTTTACATGTAAATGTCCATTTTAG.sup.77                                                                GTAAGTATTGGGTGCCCTGTCAGTGTGGGA.sup                                            .78                                   e23                                                                              5526 5586                                                                              61   TTGAATGCTCTTTCCTTCCTGGGGATCCAG.sup.79                                                                GTAAGGTGCCTCGCATGTACCTGTGCTATT.sup                                            .80                                   e24                                                                              5587 5914                                                                             328   CTAATCTCTGCTTGTGTTCTCTGTCTCCAG.sup.81                        __________________________________________________________________________     *Base numbers in SEQ ID NO: 1.                                                **Numbers in superscript refer to SEQ ID NOS.                                 ***e4 from SEQ ID NO: 11.                                                

Low stringency blots in which genomic DNA from organisms of diversephylogenetic background were probed with BRCA1 sequences that lack thezinc-finger region revealed strongly hybridizing fragments in human,monkey, sheep and pig, and very weak hybridization signals in rodents.This result indicates that, apart from the zinc-finger domain, BRCA1 isconserved only at a moderate level through evolution.

Germline BRCA1 mutations in 17q-linked kindreds. The most rigorous testfor BRCA1 candidate genes is to search for potentially disruptivemutations in carrier individuals from kindreds that segregate 17q-linkedsusceptibility to breast and ovarian cancer. Such individuals mustcontain BRCA1 alleles that differ from the wildtype sequence. The set ofDNA samples used in this analysis consisted of DNA from individualsrepresenting 8 different BRCA1 kindreds (Table 10).

                  TABLE 10                                                        ______________________________________                                        KINDRED DESCRIPTIONS AND ASSOCIATED LOD SCORES                                Cases          Sporadic                                                       (n)            Cases.sup.1                                                                            LOD                                                   Kindred                                                                             Br    Br < 50 Ov   (n)    Score Markers(s)                              ______________________________________                                        2082  31    20      22   7      9.49  D17S1327                                2099  22    14       2*  0      2.36  D17S800/D17S855.sup.2                   2035  10     8       1*  0      2.25  D17S1327                                1901  10     7       1*  0      1.50  D17S855                                 1925   4     3       0   0      0.55  D17S579                                 1910   5     4       0   0      0.36  D17S579/D17S250.sup.2                   1927   5     4       0   1      -0.44 D17S250                                 1911   8     5       0   2      -0.20 D17S250                                 ______________________________________                                         .sup.1 Number of women with breast cancer (diagnosed under age 50) or         ovarian cancer (diagnosed at any age) who do not share the BRCA1linked        haplotype segregating in the remainder of the cases in the kindred.           .sup.2 Multipoint LOD score calculated using both markers                     *kindred contains one individual who had both breast and ovarian cancer;      this individual is counted as a breast cancer case and as an ovarian          cancer case.                                                             

The logarithm of the odds (LOD) scores in these kindreds range from 9.49to -0.44 for a set of markers in 17q21. Four of the families haveconvincing LOD scores for linkage, and 4 have low positive or negativeLOD scores. The latter kindreds were included because they demonstratehaplotype sharing at chromosome 17q21 for at least 3 affected members.Furthennore, all kindreds in the set display early age of breast canceronset and 4 of the kindreds include at least one case of ovarian cancer,both hallmarks of BRCA1 kindreds. One kindred, 2082, has nearly equalincidence of breast and ovarian cancer, an unusual occurrence given therelative rarity of ovarian cancer in the population. All of the kindredsexcept two were ascertained in Utah. K2035 is from the midwest. K2099 isan African-American kindred from the southern USA.

In the initial screen for predisposing mutations in BRCA1, DNA from oneindividual who carries the perdisplotype in each kindred was tested. The23 coding exons and associated splice junction were amplified eitherfrom genomic DNA samples or from cDNA prepared from lymphocyte mRNA.When the amplified DNA sequences were compared to the wildtype sequence,4 of the 8 kindred samples were found to contain sequence variants(Table 11).

                  TABLE 11                                                        ______________________________________                                        PREDISPOSING MUTATIONS                                                        Kindred Number                                                                           Mutation    Coding Effect                                                                              Location*                                 ______________________________________                                        2082       C → T                                                                              Gln → Stop                                                                          4056                                      1910       extra C     frameshift   5385                                      2099       T → G                                                                              Met → Arg                                                                           5443                                      2035       ?           loss of transcript                                     1901       11 bp deletion                                                                            frameshift    189                                      ______________________________________                                         *In Sequence ID NO: 1                                                    

All four sequence variants are heterozygous and each appears in only oneof the kindreds. Kinderd 2082 contains a nonsense mutation in exon 11(FIG. 9A), Kindred 1910 contains a single nucleoting insertion in exon20 (FIG. 9B), and Kindred 2099 contains a missense mutation in exon 21,resulting in a Met→Arg substitution. The frameshift and nonsensemutations are likely disruptive to the function of the BRCA1 product.The peptide encoded by the frameshift allele in Kindred 1910 wouldcontain an altered amino acid sequence beginning 108 residues from thewildtype C-terminus. The peptide encoded by the frameshift allele inKindred 1901 would contain an altered amino acid sequence beginning withthe 24th residue from the wildtype N-terminus. The mutant allele inKindred 2082 would encode a protein missing 551 residues from theC-terminus. The missense substitution observed in Kindred 2099 ispotentially disruptive as it causes the replacement of a smallhydrophobic amino acid (Met), by a large charged residue (Arg). Elevencommon polymorphisms were also identified, 8 in coding sequence and 3 inintrons.

The individual studied in Kindred 2035 evidently contains a regulatorymutation in BRCA 1. In her cDNA, a polymorphic site (A→G at base 3667)appeared homozygous, whereas her genomic DNA revealed heterozygosity atthis position (FIG. 9C). A possible explanation for this observation isthat mRNA from her mutated BRCA1 allele is absent due to a mutation thataffects its production or stability. This possibility was exploredfurther by examining 5 polymorphic sites in the BRCA1 coding region,which are separated by as much as 3.5 kb in the BRCA1 transcript. In allcases where her genomic DNA appeared heterozygous for a polymorphism,cDNA appeared homozygous. In individuals from other kindreds and innon-haplotype carriers in Kindred 2035, these polymorphic sites could beobserved as heterozygous in cDNA, implying that amplification from cDNAwas not biased in favor of one allele. This analysis indicates that aBRCA1 mutation in Kindred 2035 either prevents transcription or causesinstability or aberrant splicing of the BRCA1 transcript.

Cosegregation of BRCA1 mutations with BRCA1haplotypes and populationfrequency analysis. In addition to potentially disrupting proteinfunction, two criteria must be met for a sequence variant to qualify asa candidate predisposing mutation. The variant must: 1) be present inindividuals from the kindred who carry the predisposing BRCA 1 haplotypeand absent in other members of the kindred, and 2) be rare in thegeneral population.

Each mutation was tested for cosegregation with BRCA1. For theframeshift mutation in Kindred 1910, two other haplotype carriers andone non-carrier were sequenced (FIG. 9B). Only the carriers exhibitedthe frameshift mutation. The C to T change in Kindred 2082 created a newAvrII restriction site. Other carriers and non-carriers in the kindredwere tested for the presence of the restriction site (FIG. 9A). Anallele-specific oligonucleotide (ASO) was designed to detect thepresence of the sequence variant in Kindred 2099. Several individualsfrom the kindred, some known to carry the haplotype associated with thepredisposing allele, and others known not to carry the associatedhaplotype, were screened by ASO for the mutation previously detected inthe kindred. In each kindred, the corresponding mutant allele wasdetected in individuals carrying the BRCA1-associated haplotype, and wasnot detected in noncaitiers. In the case of the potential regulatorymutation observed in the individual from Kindred 2035, cDNA and genomicDNA from carriers in the kindred were compared for heterozygosity atpolymorphic sites. In every instance, the extinguished allele in thecDNA sample was shown to lie on the chromosome that carries the BRCA1predisposing allele (FIG. 9C).

To exclude the possibility that the mutations were simply commonpolymorphisms in the population, ASOs for each mutation were used toscreen a set of normal DNA samples. Gene frequency estimates inCaucasians were based on random samples from the Utah population. Genefrequency estimates in African-Americans were based on 39 samplesprovided by M. Peracek-Vance which originate from African-Americans usedin her linkage studies and 20 newborn Utah African-Americans. None ofthe 4 potential predisposing mutations was found in the appropriatecontrol population, indicating that they are rare in the generalpopulation. Thus, two important requirements for BRCA1 susceptibilityalleles were fulfilled by the candidate predisposing mutations: 1)cosegregation of the mutant allele with disease, and 2) absence of themutant allele in controls, indicating a low gene frequency in thegeneral population.

Phenotvpic Expression of BRCA1 Mutations. The effect of the mutations onthe BRCA1 protein correlated with differences in the observed phenotypicexpression in the BRCA1 kindreds. Most BRCA 1 kindreds have a moderatelyincreased ovarian cancer risk, and a smaller subset have high risks ofovarian cancer, comparable to those for breast cancer (Easton et al.,1993). Three of the four kindreds in which BRCA1 mutations were detectedfall into the former category, while the fourth (K2082) falls into thehigh ovarian cancer risk group. Since the BRCA1 nonsense mutation foundin K2082 lies closer to the amino terminus than the other mutationsdetected, it might be expected to have a different phenotype. In fact,Kindred K2082 mutation has a high incidence of ovarian cancer, and alater mean age at diagnosis of breast cancer cases than the otherkindreds (Goldgar et al., 1994). This difference in age of onset couldbe due to an ascertainment bias in the smaller, more highly penetrantfamilies, or it could reflect tissue-specific differences in thebehavior of BRCA1 mutations. The other 3 kindreds that segregate knownBRCA1 mutations have, on average, one ovarian cancer for every 10 casesof breast cancer, but have a high proportion of breast cancer casesdiagnosed in their late 20's or early 30's. Kindred 1910, which has aframeshift mutation, is noteworthy because three of the four affectedindividuals had bilateral breast cancer, and in each case the secondtumor was diagnosed within a year of the first occurrence. Kindred 2035,which segregates a potential regulatory BRCA1 mutation, might also beexpected to have a dramatic phenotype. Eighty percent of breast cancercases in this kindred occur under age 50. This figure is as high as anyin the set, suggesting a BRCA1 mutant allele of high penetrance (Table10).

Although the mutations described above clearly are deleterious, causingbreast cancer in women at very young ages, each of the four kindredswith mutations includes at least one woman who carries the mutation wholived until age 80 without developing a malignancy. It will be of utmostimportance in the studies that follow to identify other genetic orenvironmental factors that may ameliorate the effects of BRCA1mutations.

In four of the eight putative BRCA1-linked kindreds, potentialpredisposing mutations were not found. Three of these four have LODscores for BRCA1-linked markers of less than 0.55. Thus, these kindredsmay not in reality segregate BRCA1 predisposing alleles. Alternatively,the mutations in these four kindreds may lie in regions of BRCA1 that,for example, affect the level of transcript and therefore have thus farescaped detection.

Role of BRCA1 in Cancer. Most tumor suppressor genes identified to dategive rise to protein products that are absent, nonfunctional, or reducedin function. The majority of TP53 mutations are missense; some of thesehave been shown to produce abnormal p53 molecules that interfere withthe function of the wildtype product (Shaulian el al., 1992; Srivastavaet aL, 1993). A similar dominant negative mechanism of action has beenproposed for some adenomatous polyposis coli (APC) alleles that producetruncated molecules (Su et al., 1993), and for point mutations in theWilms' tumor gene (WT1) that alter DNA binding of the protein (Little etal., 1993). The nature of the mutations observed in the BRCA1 codingsequence is consistent with production of either dominant negativeproteins or nonfunctional proteins. The regulatory mutation inferred inKindred 2035 cannot be a dominant negative; rather, this mutation likelycauses reduction or complete loss of BRCA1 expression from the affectedallele.

The BRCA1 protein contains a C₃ HC₄ zinc-finger domain, similar to thosefound in numerous DNA binding proteins and implicated in zinc-dependentbinding to nucleic acids. The first 180 amino acids of BRCA1 containfive more basic residues than acidic residues. In contrast, theremainder of the molecule is very acidic, with a net excess of 70 acidicresidues. The excess negative charge is particularly concentrated nearthe C-terminus. Thus, one possibility is that BRCA1 encodes atranscription factor with an N-terminal DNA binding domain and aC-terminal transactivational "acidic blob" domain. Interestingly,another familial tumor suppressor gene, WT1, also contains a zinc-fingermotif (Haber el al., 1990). Many cancer predisposing mutations in WT1alter zinc-finger domains (Little et al., 1993; Haber et al., 1990;Little et al., 1992). WT1 encodes a transcription factor, andalternative splicing of exons that encode parts of the zinc-fingerdomain alter the DNA binding properties of WT1 (Bickmore et al., 1992).Some alternatively spliced forms of WT1 mRNA generate molecules that actas transcriptional repressors (Drummond et al., 1994). Some BRCA1splicing variants may alter the zinc-finger motif, raising thepossibility that a regulatory mechanism similar to 10 that which occursin WT1 may apply to BRCA1.

EXAMPLE 9 Analysis of Tumors for BRCA1 Mutations

To focus the analysis on tumors most likely to contain BRCA1 mutations,primary breast and ovarian carcinomas were typed for LOH in the BRCA1region. Three highly polymorphic, simple tandem repeat markers were usedto assess LOH: D17S1323 and D17S855, which are intragenic to BRCA1, andD17S1327, which lies approximately 100 kb distal to BRCA1 . The combinedLOH frequency in informative cases (i.e., where the germline washeterozygous) was 32/72 (44%) for the breast carcinomas and 12/21 (57%)for the ovarian carcinomas, consistent with previous measurements of LOHin the region (Futreal et al., 1992b; Jacobs et al., 1993; Sato et al.,1990; Eccles et al., 1990; Cropp et aL, 1994). The analysis thus defineda panel of 32 breast tumors and 12 ovarian tumors of mixed race and ageof onset to be examined for BRCA mutations. The complete 5,589 bp codingregion and intron/exon boundary sequences of the gene were screened inthis tumor set by direct sequencing alone or by a combination ofsingle-strand conformation analysis (SSCA) and direct sequencing.

A total of six mutations (of which two are identical) was found, one inan ovarian tumor, four in breast tumors and one in a male unaffectedhaplotype carrier (Table 12). One mutation, Glu 1541Ter, introduced astop codon that would create a truncated protein missing 323 amino acidsat the carboxy terminus. In addition, two missense mutations wereidentified. These are Ala1708Glu and Met1775Arg and involvesubstitutions of small, hydrophobic residues by charged residues.Patients 17764 and 19964 are from the same family. In patient OV24nucleotide 2575 is deleted and in patients 17764 and 19964 nucleotides2993-2996 are deleted.

                  TABLE 12                                                        ______________________________________                                        Predisposing Mutations                                                                       Nucleotide Amino Acid                                                                            Age of Family                               Patient                                                                             Codon    Change     Change  Onset  History                              ______________________________________                                        BT098 1541     GAG → TAG                                                                         Glu → Stop                                                                     39     -                                    OV24   819     1 bp deletion                                                                            frameshift                                                                            44     -                                    BT106 1708     GCG → GAG                                                                         Ala → Glu                                                                      24     +                                    MC44  1775     ATG → AGG                                                                         Met → Arg                                                                      42     +                                    17764  958     4 bp deletion                                                                            frameshift                                                                            31     +                                    19964  958     4 bp deletion                                                                            frameshift      +*                                  ______________________________________                                         *Unaffected haplotype carrier, male                                      

Several lines of evidence suggest that all five mutations representBRCA1 susceptibility alleles:

(i) all mutations are present in the germline;

(ii) all are absent in appropriate control populations, suggesting theyare not common polymorphisms;

(iii) each mutant allele is retained in the tumor, as is the case intumors from patients belonging to kindreds that segregate BRCA1susceptibility alleles (Smith et al., 1992; Kelsell et al., 1993) (ifthe mutations represented neutral polymorphisms, they should be retainedin only 50% of the cases);

(iv) the age of onset in the four breast cancer cases with mutationsvaried between 24 and 42 years of age, consistent with the early age ofonset of breast cancer in individuals with BRCA1 susceptibility;similarly, the ovarian cancer case was diagnosed at 44, an age thatfalls in the youngest 13% of all ovarian cancer cases; and finally,

(v) three of the five cases have positive family histories of breast orovarian cancer found retrospectively in their medical records, althoughthe tumor set was not selected with regard to this criterion.

BT106 was diagnosed at a very early age with breast cancer. Her motherhad ovarian cancer, her father had melanoma, and her paternalgrandmother also had breast cancer. Patient MC44, an African-American,had bilateral breast cancer at an early age. This patient had a sisterwho died of breast cancer at a very early age. Her mutation (Met1775Arg)had been detected previously in Kindred 2099, an African-American familythat segregates a BRCA1 susceptibility allele, and was absent inAfrican-American and Caucasian controls.

Patient MC44, to our knowledge, is unrelated to Kindred 2099. Thedetection of a rare mutant allele, once in a BRCA1 kindred and once inthe germline of an apparently unrelated early-onset breast cancer case,suggests that the Met1775Arg change may be a common predisposingmutation in African-Americans. Collectively, these observations indicatethat all four BRCA1 mutations in tumors represent susceptibilityalleles; no somatic mutations were detected in the samples analyzed.

The paucity of somatic BRCA1 mutations is unexpected, given thefrequency of LOH on 17q, and the usual role of susceptibility genes astumor suppressors in cancer progression. There are three possibleexplanations for this result: (i) some BRCA1 mutations in codingsequences were missed by our screening procedure; (ii) BRCA1 somaticmutations fall primarily outside the coding exons; and (iii) LOH eventsin 17q do not reflect BRCA1 somatic mutations.

If somatic BRCA1 mutations truly are rare in breast and ovarycarcinomas, this would have strong implications for the biology ofBRCA1. The apparent lack of somatic BRCA1 mutations implies that theremay be some fundamental difference in the genesis of tumors ingenetically predisposed BRCA1 carriers, compared with tumors in thegeneral population. For example, mutations in BRCA1 may have an effectonly on tumor foination at a specific stage early in breast and ovariandevelopment. This possibility is consistent with a primary function forBRCA1 in premenopausal breast cancer. Such a model for the role of BRCA1in breast and ovarian cancer predicts an interaction betweenreproductive hormones and BRCA1 function. However, no clinical orpathological differences in familial versus sporadic breast and ovarytumors, other than age of onset, have been described (Lynch et al.,1990). On the other hand, the recent finding of increased TP53 mutationand microsatellite instability in breast tumors from patients with afamily history of breast cancer (Glebov et al., 1994) may reflect somedifference in tumors that arise in genetically predisposed persons. Theinvolvement of BRCA1 in this phenomenon can now be addressed directly.Alternatively, the lack of somatic BRCA1 mutations may result from theexistence of multiple genes that function in the same pathway of tumorsuppression as BRCA1, but which collectively represent a more favoredtarget for mutation in sporadic tumors. Since mutation of a singleelement in a genetic pathway is generally sufficient to disrupt thepathway, BRCA1 might mutate at a rate that is far lower than the sum ofthe mutational rates of the other elements.

EXAMPLE 10 Analysis of the BRCA1 Gene

The structure and function of BRCA1 gene are determined according to thefollowing methods.

Biological Studies. Mammalian expression vectors containing BRCA1 cDNAare constructed and transfected into appropriate breast carcinoma cellswith lesions in the gene. Wild-type BRCA1 cDNA as well as altered BRCA1cDNA are utilized. The altered BRCA1 cDNA can be obtained from alteredBRCA1 alleles or produced as described below. Phenotypic reversion incultures (e.g., cell morphology, doubling time, anchorage-independentgrowth) and in animals (e.g., tumorigenicity) is examined. The studieswill employ both wild-type and mutant forms (Section B) of the gene.

Molecular Genetics Studies. In vitro mutagenesis is performed toconstruct deletion mutants and missense mutants (by single base-pairsubstitutions in individual codons and cluster charged→alanine scanningmutagenesis). The mutants are used in biological, biochemical andbiophysical studies.

Mechanism Studies. The ability of BRCA1 protein to bind to known andunknown DNA sequences is examined. Its ability to transactivatepromoters is analyzed by transient reporter expression systems inmammalian cells. Conventional procedures such as particle-capture andyeast two-hybrid system are used to discover and identify any functionalpartners. The nature and functions of the partners are characterized.These partners in turn are targets for drug discovery.

Structural Studies. Recombinant proteins are produced in E. coli, yeast,insect and/or mammalian cells and are used in crystallographical and NMRstudies. Molecular modeling of the proteins is also employed. Thesestudies facilitate structure-driven drug design.

EXAMPLE 11 Two Step Assay to Detect the Presence of BRCA1 in a Sample

Patient sample is processed according to the method disclosed byAntonarakis et al. (1985), separated through a 1% agarose gel andtransferred to nylon membrane for Southern blot analysis.

Membranes are UV cross linked at 150 mJ using a GS Gene Linker(Bio-Rad). BRCA1 probe corresponding to nucleotide positions 3631-3930of SEQ ID NO:1 is subcloned into pTZ18U. The phagemids are transformedinto E. coli MV1190 infected with M13KO7 helper phage (Bio-Rad,Richmond, Calif.). Single stranded DNA is isolated according to standardprocedures (see Sambrook et al., 1989).

Blots are prehybridized for 15-30 min at 65° C. in 7% sodium dodecylsulfate (SDS) in 0.5M NaPO₄. The methods follow those described byNguyen et al., 1992. The blots are hybridized overnight at 65° C. in 7%SDS, 0.5M NaPO₄ with 25-50 ng/ml single stranded probe DNA.Post-hybridization washes consist of two 30 min washes in 5% SDS, 40 mMNaPO₄ at 65° C., followed by two 30 min washes in 1% SDS, 40 mM NaPO₄ at65° C.

Next the blots are rinsed with phosphate buffered saline (pH 6.8) for 5min at room temperature and incubated with 0.2% casein in PBS for 30-60min at room temperature and rinsed in PBS for 5 min. The blots are thenpreincubated for 5-10 minutes in a shaking water bath at 45° C. withhybridization buffer consisting of 6M urea, 0.3M NaCI, and 5X Denhardt'ssolution (see Sambrook, et al., 1989). The buffer is removed andreplaced with 50-75 μl/cm² fresh hybridization buffer plus 2.5 nM of thecovalently cross-linked oligonucleotide-alkaline phosphatase conjugatewith the nucleotide sequence complementary to the universal primer site(UP-AP, Bio-Rad). The blots are hybridized for 20-30 min at 45° C. andpost hybridization washes are incubated at 45° C. as two 10 min washesin 6M urea, 1× standard saline citrate (SSC), 0.1% SDS and one 10 minwash in 1× SSC, 0.1% Triton®X-100. The blots are rinsed for 10 min atroom temperature with 1× SSC.

Blots are incubated for 10 min at room temperature with shaking in thesubstrate buffer consisting of 0.1M diethanolamine, 1 mM MgCI₂, 0.02%sodium azide, pH 10.0. Individual blots are placed in heat sealable bagswith substrate buffer and 0.2 mM AMPPD (3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane, disodium salt, Bio-Rad).After a 20 min incubation at room temperature with shaking, the excessAMPPD solution is removed. The blot is exposed to X-ray film overnight.Positive bands indicate the presence of BRCA1.

EXAMPLE 12 Generation of Polyclonal Antibody against BRCA1

Segments of BRCA1 coding sequence were expressed as fusion protein in E.coli. The overexpressed protein was purified by gel elution and used toimmunize rabbits and mice using a procedure similar to the one describedby Harlow and Lane, 1988. This procedure has been shown to generate Absagainst various other proteins (for example, see Kraemer et al., 1993).

Briefly, a stretch of BRCA1 coding sequence was cloned as a fusionprotein in plasmid PET5A (Novagen, Inc., Madison, Wis.). The BRCA1incorporated sequence includes the amino acids corresponding to#1361-1554 of SEQ ID NO:2. After induction with IPTG, the overexpressionof a fusion protein with the expected molecular weight was verified bySDS/PAGE. Fusion protein was purified from the gel by electroelution.The identification of the protein as the BRCA1 fusion product wasverified by protein sequencing at the N-terminus. Next, the purifiedprotein was used as immunogen in rabbits. Rabbits were immunized with100 μg of the protein in complete Freund's adjuvant and boosted twice in3 week intervals, first with 100μg of immunogen in incomplete Freund'sadjuvant followed by 100 μg of immunogen in PBS. Antibody containingserum is collected two weeks thereafter.

This procedure is repeated to generate antibodies against the mutantforms of the BRCA1 gene.

These antibodies, in conjunction with antibodies to wild type BRCA1, areused to detect the presence and the relative level of the mutant formsin various tissues and biological fluids. EXAMPILE 13

Generation of Monoclonal Antibodies Specific for BRCA1

Monoclonal antibodies are generated according to the following protocol.Mice are immunized with immunogen comprising intact BRCA1 or BRCA1peptides (wild type or mutant) conjugated to keyhole limpet hemocyaninusing glutaraldehyde or EDC as is well known.

The immunogen is mixed with an adjuvant. Each mouse receives fourinjections of 10 to 100 μg of immunogen and after the fourth injectionblood samples are taken from the mice to determine if the serum containsantibody to the immunogen. Serum titer is determined by ELISA or RIA.Mice with sera indicating the presence of antibody to the immunogen areselected for hybridoma production.

Spleens are removed from immune mice and a single cell suspension isprepared (see Harlow and Lane, 1988). Cell fuisions are performedessentially as described by Kohler and Milstein, 1975. Briefly, P3.65.3myeloma cells (American Type Culture Collection, Rockville, Md.) arefused with immune spleen cells using polyethylene glycol as described byHarlow and Lane, 1988. Cells are plated at a density of 2×10⁵ cells/wellin 96 well tissue culture plates. Individual wells are examined forgrowth and the supernatants of wells with growth are tested for thepresence of BRCA1 specific antibodies by ELISA or RIA using wild type ormutant BRCA1 target protein. Cells in positive wells are expanded andsubcloned to establish and confirm monoclonality.

Clones with the desired specificities are expanded and grown as ascitesin mice or in a hollow fiber system to produce sufficient quantities ofantibody for characterization and assay development.

EXAMPLE 14 Sandwich Assay for BRCA1

Monoclonal antibody is attached to a solid surface such as a plate,tube, bead, or particle. Preferably, the antibody is attached to thewell surface of a 96-well ELISA plate. 100 μl sample (e.g., serum,urine, tissue cytosol) containing the BRCA1 peptide/protein (wild-typeor mutant) is added to the solid phase antibody. The sample is incubatedfor 2 hrs at room temperature. Next the sample fluid is decanted, andthe solid phase is washed with buffer to remove tunbound material. 100μl of a second monoclonal antibody (to a different determinant on theBRCA1 peptide/protein) is added to the solid phase. This antibody islabeled with a detector molecule (e.g., ¹²⁵ I, enzyme, fluorophore, or achromophore) and the solid phase with the second antibody is incubatedfor two hrs at room temperature. The second antibody is decanted and thesolid phase is washed with buffer to remove unbound material.

The amount of bound label, which is proportional to the amount of BRCA1peptide/protein present in the sample, is quantitated. Separate assaysare performed using monoclonal antibodies which are specific for thewild-type BRCA1 as well as monoclonal antibodies specific for each ofthe mutations identified in BRCA1.

Industrial Utility

As previously described above, the present invention provides materialsand methods for use in testing BRCA1 alleles of an individual and aninterpretation of the normal or predisposing nature of the alleles.Individuals at higher than normal risk might modify their lifestylesappropriately. In the case of BRCA1, the most significant non-geneticrisk factor is the protective effect of an early, full term pregnancy.Therefore, women at risk could consider early childbearing or a therapydesigned to simulate the hormonal effects of an early full-termpregnancy. Women at high risk would also strive for early detection andwould be more highly motivated to learn and practice breast selfexamination. Such women would also be highly motivated to have regularmammograms, perhaps starting at an earlier age than the generalpopulation. Ovarian screening could also be undertaken at greaterfrequency. Diagnostic methods based on sequence analysis of the BRCA1locus could also be applied to tumor detection and classification.Sequence analysis could be used to diagnose precursor lesions. With theevolution of the method and the accumulation of information about BRCA1and other causative loci, it could become possible to separate cancersinto benign and malignant.

Women with breast cancers may follow different surgical procedures ifthey are predisposed, and therefore likely to have additional cancers,than if they are not predisposed. Other therapies may be developed,using either peptides or small molecules (rational drug design).Peptides could be the missing gene product itself or a portion of themissing gene product. Alternatively, the therapeutic agent could beanother molecule that mimics the deleterious gene's function, either apeptide or a nonpeptidic molecule that seeks to counteract thedeleterious effect of the inherited locus. The therapy could also begene based, through introduction of a normal BRCA1 allele intoindividuals to make a protein which will counteract the effect of thedeleterious allele. These gene therapies may take many forms and may bedirected either toward preventing the tumor from forming, curing acancer once it has occurred, or stopping a cancer from metastasizing.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

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Hitzeman et al., EP 73,675A

PCT published application WO 93/07282

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 85                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5914 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 120..5708                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AGCTCGCTGAGACTTCCTGGACCCCGCACCAGGCTGTGGGGTTTCTCAGATAACTGGGCC60                CCTGCGCTCAGGAGGCCTTCACCCTCTGCTCTGGGTAAAGTTCATTGGAACAGAAAGAA119                ATGGATTTATCTGCTCTTCGCGTTGAAGAAGTACAAAATGTCATTAAT167                           MetAspLeuSerAlaLeuArgValGluGluValGlnAsnValIleAsn                              151015                                                                        GCTATGCAGAAAATCTTAGAGTGTCCCATCTGTCTGGAGTTGATCAAG215                           AlaMetGlnLysIleLeuGluCysProIleCysLeuGluLeuIleLys                              202530                                                                        GAACCTGTCTCCACAAAGTGTGACCACATATTTTGCAAATTTTGCATG263                           GluProValSerThrLysCysAspHisIlePheCysLysPheCysMet                              354045                                                                        CTGAAACTTCTCAACCAGAAGAAAGGGCCTTCACAGTGTCCTTTATGT311                           LeuLysLeuLeuAsnGlnLysLysGlyProSerGlnCysProLeuCys                              505560                                                                        AAGAATGATATAACCAAAAGGAGCCTACAAGAAAGTACGAGATTTAGT359                           LysAsnAspIleThrLysArgSerLeuGlnGluSerThrArgPheSer                              65707580                                                                      CAACTTGTTGAAGAGCTATTGAAAATCATTTGTGCTTTTCAGCTTGAC407                           GlnLeuValGluGluLeuLeuLysIleIleCysAlaPheGlnLeuAsp                              859095                                                                        ACAGGTTTGGAGTATGCAAACAGCTATAATTTTGCAAAAAAGGAAAAT455                           ThrGlyLeuGluTyrAlaAsnSerTyrAsnPheAlaLysLysGluAsn                              100105110                                                                     AACTCTCCTGAACATCTAAAAGATGAAGTTTCTATCATCCAAAGTATG503                           AsnSerProGluHisLeuLysAspGluValSerIleIleGlnSerMet                              115120125                                                                     GGCTACAGAAACCGTGCCAAAAGACTTCTACAGAGTGAACCCGAAAAT551                           GlyTyrArgAsnArgAlaLysArgLeuLeuGlnSerGluProGluAsn                              130135140                                                                     CCTTCCTTGCAGGAAACCAGTCTCAGTGTCCAACTCTCTAACCTTGGA599                           ProSerLeuGlnGluThrSerLeuSerValGlnLeuSerAsnLeuGly                              145150155160                                                                  ACTGTGAGAACTCTGAGGACAAAGCAGCGGATACAACCTCAAAAGACG647                           ThrValArgThrLeuArgThrLysGlnArgIleGlnProGlnLysThr                              165170175                                                                     TCTGTCTACATTGAATTGGGATCTGATTCTTCTGAAGATACCGTTAAT695                           SerValTyrIleGluLeuGlySerAspSerSerGluAspThrValAsn                              180185190                                                                     AAGGCAACTTATTGCAGTGTGGGAGATCAAGAATTGTTACAAATCACC743                           LysAlaThrTyrCysSerValGlyAspGlnGluLeuLeuGlnIleThr                              195200205                                                                     CCTCAAGGAACCAGGGATGAAATCAGTTTGGATTCTGCAAAAAAGGCT791                           ProGlnGlyThrArgAspGluIleSerLeuAspSerAlaLysLysAla                              210215220                                                                     GCTTGTGAATTTTCTGAGACGGATGTAACAAATACTGAACATCATCAA839                           AlaCysGluPheSerGluThrAspValThrAsnThrGluHisHisGln                              225230235240                                                                  CCCAGTAATAATGATTTGAACACCACTGAGAAGCGTGCAGCTGAGAGG887                           ProSerAsnAsnAspLeuAsnThrThrGluLysArgAlaAlaGluArg                              245250255                                                                     CATCCAGAAAAGTATCAGGGTAGTTCTGTTTCAAACTTGCATGTGGAG935                           HisProGluLysTyrGlnGlySerSerValSerAsnLeuHisValGlu                              260265270                                                                     CCATGTGGCACAAATACTCATGCCAGCTCATTACAGCATGAGAACAGC983                           ProCysGlyThrAsnThrHisAlaSerSerLeuGlnHisGluAsnSer                              275280285                                                                     AGTTTATTACTCACTAAAGACAGAATGAATGTAGAAAAGGCTGAATTC1031                          SerLeuLeuLeuThrLysAspArgMetAsnValGluLysAlaGluPhe                              290295300                                                                     TGTAATAAAAGCAAACAGCCTGGCTTAGCAAGGAGCCAACATAACAGA1079                          CysAsnLysSerLysGlnProGlyLeuAlaArgSerGlnHisAsnArg                              305310315320                                                                  TGGGCTGGAAGTAAGGAAACATGTAATGATAGGCGGACTCCCAGCACA1127                          TrpAlaGlySerLysGluThrCysAsnAspArgArgThrProSerThr                              325330335                                                                     GAAAAAAAGGTAGATCTGAATGCTGATCCCCTGTGTGAGAGAAAAGAA1175                          GluLysLysValAspLeuAsnAlaAspProLeuCysGluArgLysGlu                              340345350                                                                     TGGAATAAGCAGAAACTGCCATGCTCAGAGAATCCTAGAGATACTGAA1223                          TrpAsnLysGlnLysLeuProCysSerGluAsnProArgAspThrGlu                              355360365                                                                     GATGTTCCTTGGATAACACTAAATAGCAGCATTCAGAAAGTTAATGAG1271                          AspValProTrpIleThrLeuAsnSerSerIleGlnLysValAsnGlu                              370375380                                                                     TGGTTTTCCAGAAGTGATGAACTGTTAGGTTCTGATGACTCACATGAT1319                          TrpPheSerArgSerAspGluLeuLeuGlySerAspAspSerHisAsp                              385390395400                                                                  GGGGAGTCTGAATCAAATGCCAAAGTAGCTGATGTATTGGACGTTCTA1367                          GlyGluSerGluSerAsnAlaLysValAlaAspValLeuAspValLeu                              405410415                                                                     AATGAGGTAGATGAATATTCTGGTTCTTCAGAGAAAATAGACTTACTG1415                          AsnGluValAspGluTyrSerGlySerSerGluLysIleAspLeuLeu                              420425430                                                                     GCCAGTGATCCTCATGAGGCTTTAATATGTAAAAGTGAAAGAGTTCAC1463                          AlaSerAspProHisGluAlaLeuIleCysLysSerGluArgValHis                              435440445                                                                     TCCAAATCAGTAGAGAGTAATATTGAAGACAAAATATTTGGGAAAACC1511                          SerLysSerValGluSerAsnIleGluAspLysIlePheGlyLysThr                              450455460                                                                     TATCGGAAGAAGGCAAGCCTCCCCAACTTAAGCCATGTAACTGAAAAT1559                          TyrArgLysLysAlaSerLeuProAsnLeuSerHisValThrGluAsn                              465470475480                                                                  CTAATTATAGGAGCATTTGTTACTGAGCCACAGATAATACAAGAGCGT1607                          LeuIleIleGlyAlaPheValThrGluProGlnIleIleGlnGluArg                              485490495                                                                     CCCCTCACAAATAAATTAAAGCGTAAAAGGAGACCTACATCAGGCCTT1655                          ProLeuThrAsnLysLeuLysArgLysArgArgProThrSerGlyLeu                              500505510                                                                     CATCCTGAGGATTTTATCAAGAAAGCAGATTTGGCAGTTCAAAAGACT1703                          HisProGluAspPheIleLysLysAlaAspLeuAlaValGlnLysThr                              515520525                                                                     CCTGAAATGATAAATCAGGGAACTAACCAAACGGAGCAGAATGGTCAA1751                          ProGluMetIleAsnGlnGlyThrAsnGlnThrGluGlnAsnGlyGln                              530535540                                                                     GTGATGAATATTACTAATAGTGGTCATGAGAATAAAACAAAAGGTGAT1799                          ValMetAsnIleThrAsnSerGlyHisGluAsnLysThrLysGlyAsp                              545550555560                                                                  TCTATTCAGAATGAGAAAAATCCTAACCCAATAGAATCACTCGAAAAA1847                          SerIleGlnAsnGluLysAsnProAsnProIleGluSerLeuGluLys                              565570575                                                                     GAATCTGCTTTCAAAACGAAAGCTGAACCTATAAGCAGCAGTATAAGC1895                          GluSerAlaPheLysThrLysAlaGluProIleSerSerSerIleSer                              580585590                                                                     AATATGGAACTCGAATTAAATATCCACAATTCAAAAGCACCTAAAAAG1943                          AsnMetGluLeuGluLeuAsnIleHisAsnSerLysAlaProLysLys                              595600605                                                                     AATAGGCTGAGGAGGAAGTCTTCTACCAGGCATATTCATGCGCTTGAA1991                          AsnArgLeuArgArgLysSerSerThrArgHisIleHisAlaLeuGlu                              610615620                                                                     CTAGTAGTCAGTAGAAATCTAAGCCCACCTAATTGTACTGAATTGCAA2039                          LeuValValSerArgAsnLeuSerProProAsnCysThrGluLeuGln                              625630635640                                                                  ATTGATAGTTGTTCTAGCAGTGAAGAGATAAAGAAAAAAAAGTACAAC2087                          IleAspSerCysSerSerSerGluGluIleLysLysLysLysTyrAsn                              645650655                                                                     CAAATGCCAGTCAGGCACAGCAGAAACCTACAACTCATGGAAGGTAAA2135                          GlnMetProValArgHisSerArgAsnLeuGlnLeuMetGluGlyLys                              660665670                                                                     GAACCTGCAACTGGAGCCAAGAAGAGTAACAAGCCAAATGAACAGACA2183                          GluProAlaThrGlyAlaLysLysSerAsnLysProAsnGluGlnThr                              675680685                                                                     AGTAAAAGACATGACAGCGATACTTTCCCAGAGCTGAAGTTAACAAAT2231                          SerLysArgHisAspSerAspThrPheProGluLeuLysLeuThrAsn                              690695700                                                                     GCACCTGGTTCTTTTACTAAGTGTTCAAATACCAGTGAACTTAAAGAA2279                          AlaProGlySerPheThrLysCysSerAsnThrSerGluLeuLysGlu                              705710715720                                                                  TTTGTCAATCCTAGCCTTCCAAGAGAAGAAAAAGAAGAGAAACTAGAA2327                          PheValAsnProSerLeuProArgGluGluLysGluGluLysLeuGlu                              725730735                                                                     ACAGTTAAAGTGTCTAATAATGCTGAAGACCCCAAAGATCTCATGTTA2375                          ThrValLysValSerAsnAsnAlaGluAspProLysAspLeuMetLeu                              740745750                                                                     AGTGGAGAAAGGGTTTTGCAAACTGAAAGATCTGTAGAGAGTAGCAGT2423                          SerGlyGluArgValLeuGlnThrGluArgSerValGluSerSerSer                              755760765                                                                     ATTTCATTGGTACCTGGTACTGATTATGGCACTCAGGAAAGTATCTCG2471                          IleSerLeuValProGlyThrAspTyrGlyThrGlnGluSerIleSer                              770775780                                                                     TTACTGGAAGTTAGCACTCTAGGGAAGGCAAAAACAGAACCAAATAAA2519                          LeuLeuGluValSerThrLeuGlyLysAlaLysThrGluProAsnLys                              785790795800                                                                  TGTGTGAGTCAGTGTGCAGCATTTGAAAACCCCAAGGGACTAATTCAT2567                          CysValSerGlnCysAlaAlaPheGluAsnProLysGlyLeuIleHis                              805810815                                                                     GGTTGTTCCAAAGATAATAGAAATGACACAGAAGGCTTTAAGTATCCA2615                          GlyCysSerLysAspAsnArgAsnAspThrGluGlyPheLysTyrPro                              820825830                                                                     TTGGGACATGAAGTTAACCACAGTCGGGAAACAAGCATAGAAATGGAA2663                          LeuGlyHisGluValAsnHisSerArgGluThrSerIleGluMetGlu                              835840845                                                                     GAAAGTGAACTTGATGCTCAGTATTTGCAGAATACATTCAAGGTTTCA2711                          GluSerGluLeuAspAlaGlnTyrLeuGlnAsnThrPheLysValSer                              850855860                                                                     AAGCGCCAGTCATTTGCTCCGTTTTCAAATCCAGGAAATGCAGAAGAG2759                          LysArgGlnSerPheAlaProPheSerAsnProGlyAsnAlaGluGlu                              865870875880                                                                  GAATGTGCAACATTCTCTGCCCACTCTGGGTCCTTAAAGAAACAAAGT2807                          GluCysAlaThrPheSerAlaHisSerGlySerLeuLysLysGlnSer                              885890895                                                                     CCAAAAGTCACTTTTGAATGTGAACAAAAGGAAGAAAATCAAGGAAAG2855                          ProLysValThrPheGluCysGluGlnLysGluGluAsnGlnGlyLys                              900905910                                                                     AATGAGTCTAATATCAAGCCTGTACAGACAGTTAATATCACTGCAGGC2903                          AsnGluSerAsnIleLysProValGlnThrValAsnIleThrAlaGly                              915920925                                                                     TTTCCTGTGGTTGGTCAGAAAGATAAGCCAGTTGATAATGCCAAATGT2951                          PheProValValGlyGlnLysAspLysProValAspAsnAlaLysCys                              930935940                                                                     AGTATCAAAGGAGGCTCTAGGTTTTGTCTATCATCTCAGTTCAGAGGC2999                          SerIleLysGlyGlySerArgPheCysLeuSerSerGlnPheArgGly                              945950955960                                                                  AACGAAACTGGACTCATTACTCCAAATAAACATGGACTTTTACAAAAC3047                          AsnGluThrGlyLeuIleThrProAsnLysHisGlyLeuLeuGlnAsn                              965970975                                                                     CCATATCGTATACCACCACTTTTTCCCATCAAGTCATTTGTTAAAACT3095                          ProTyrArgIleProProLeuPheProIleLysSerPheValLysThr                              980985990                                                                     AAATGTAAGAAAAATCTGCTAGAGGAAAACTTTGAGGAACATTCAATG3143                          LysCysLysLysAsnLeuLeuGluGluAsnPheGluGluHisSerMet                              99510001005                                                                   TCACCTGAAAGAGAAATGGGAAATGAGAACATTCCAAGTACAGTGAGC3191                          SerProGluArgGluMetGlyAsnGluAsnIleProSerThrValSer                              101010151020                                                                  ACAATTAGCCGTAATAACATTAGAGAAAATGTTTTTAAAGAAGCCAGC3239                          ThrIleSerArgAsnAsnIleArgGluAsnValPheLysGluAlaSer                              1025103010351040                                                              TCAAGCAATATTAATGAAGTAGGTTCCAGTACTAATGAAGTGGGCTCC3287                          SerSerAsnIleAsnGluValGlySerSerThrAsnGluValGlySer                              104510501055                                                                  AGTATTAATGAAATAGGTTCCAGTGATGAAAACATTCAAGCAGAACTA3335                          SerIleAsnGluIleGlySerSerAspGluAsnIleGlnAlaGluLeu                              106010651070                                                                  GGTAGAAACAGAGGGCCAAAATTGAATGCTATGCTTAGATTAGGGGTT3383                          GlyArgAsnArgGlyProLysLeuAsnAlaMetLeuArgLeuGlyVal                              107510801085                                                                  TTGCAACCTGAGGTCTATAAACAAAGTCTTCCTGGAAGTAATTGTAAG3431                          LeuGlnProGluValTyrLysGlnSerLeuProGlySerAsnCysLys                              109010951100                                                                  CATCCTGAAATAAAAAAGCAAGAATATGAAGAAGTAGTTCAGACTGTT3479                          HisProGluIleLysLysGlnGluTyrGluGluValValGlnThrVal                              1105111011151120                                                              AATACAGATTTCTCTCCATATCTGATTTCAGATAACTTAGAACAGCCT3527                          AsnThrAspPheSerProTyrLeuIleSerAspAsnLeuGluGlnPro                              112511301135                                                                  ATGGGAAGTAGTCATGCATCTCAGGTTTGTTCTGAGACACCTGATGAC3575                          MetGlySerSerHisAlaSerGlnValCysSerGluThrProAspAsp                              114011451150                                                                  CTGTTAGATGATGGTGAAATAAAGGAAGATACTAGTTTTGCTGAAAAT3623                          LeuLeuAspAspGlyGluIleLysGluAspThrSerPheAlaGluAsn                              115511601165                                                                  GACATTAAGGAAAGTTCTGCTGTTTTTAGCAAAAGCGTCCAGAAAGGA3671                          AspIleLysGluSerSerAlaValPheSerLysSerValGlnLysGly                              117011751180                                                                  GAGCTTAGCAGGAGTCCTAGCCCTTTCACCCATACACATTTGGCTCAG3719                          GluLeuSerArgSerProSerProPheThrHisThrHisLeuAlaGln                              1185119011951200                                                              GGTTACCGAAGAGGGGCCAAGAAATTAGAGTCCTCAGAAGAGAACTTA3767                          GlyTyrArgArgGlyAlaLysLysLeuGluSerSerGluGluAsnLeu                              120512101215                                                                  TCTAGTGAGGATGAAGAGCTTCCCTGCTTCCAACACTTGTTATTTGGT3815                          SerSerGluAspGluGluLeuProCysPheGlnHisLeuLeuPheGly                              122012251230                                                                  AAAGTAAACAATATACCTTCTCAGTCTACTAGGCATAGCACCGTTGCT3863                          LysValAsnAsnIleProSerGlnSerThrArgHisSerThrValAla                              123512401245                                                                  ACCGAGTGTCTGTCTAAGAACACAGAGGAGAATTTATTATCATTGAAG3911                          ThrGluCysLeuSerLysAsnThrGluGluAsnLeuLeuSerLeuLys                              125012551260                                                                  AATAGCTTAAATGACTGCAGTAACCAGGTAATATTGGCAAAGGCATCT3959                          AsnSerLeuAsnAspCysSerAsnGlnValIleLeuAlaLysAlaSer                              1265127012751280                                                              CAGGAACATCACCTTAGTGAGGAAACAAAATGTTCTGCTAGCTTGTTT4007                          GlnGluHisHisLeuSerGluGluThrLysCysSerAlaSerLeuPhe                              128512901295                                                                  TCTTCACAGTGCAGTGAATTGGAAGACTTGACTGCAAATACAAACACC4055                          SerSerGlnCysSerGluLeuGluAspLeuThrAlaAsnThrAsnThr                              130013051310                                                                  CAGGATCCTTTCTTGATTGGTTCTTCCAAACAAATGAGGCATCAGTCT4103                          GlnAspProPheLeuIleGlySerSerLysGlnMetArgHisGlnSer                              131513201325                                                                  GAAAGCCAGGGAGTTGGTCTGAGTGACAAGGAATTGGTTTCAGATGAT4151                          GluSerGlnGlyValGlyLeuSerAspLysGluLeuValSerAspAsp                              133013351340                                                                  GAAGAAAGAGGAACGGGCTTGGAAGAAAATAATCAAGAAGAGCAAAGC4199                          GluGluArgGlyThrGlyLeuGluGluAsnAsnGlnGluGluGlnSer                              1345135013551360                                                              ATGGATTCAAACTTAGGTGAAGCAGCATCTGGGTGTGAGAGTGAAACA4247                          MetAspSerAsnLeuGlyGluAlaAlaSerGlyCysGluSerGluThr                              136513701375                                                                  AGCGTCTCTGAAGACTGCTCAGGGCTATCCTCTCAGAGTGACATTTTA4295                          SerValSerGluAspCysSerGlyLeuSerSerGlnSerAspIleLeu                              138013851390                                                                  ACCACTCAGCAGAGGGATACCATGCAACATAACCTGATAAAGCTCCAG4343                          ThrThrGlnGlnArgAspThrMetGlnHisAsnLeuIleLysLeuGln                              139514001405                                                                  CAGGAAATGGCTGAACTAGAAGCTGTGTTAGAACAGCATGGGAGCCAG4391                          GlnGluMetAlaGluLeuGluAlaValLeuGluGlnHisGlySerGln                              141014151420                                                                  CCTTCTAACAGCTACCCTTCCATCATAAGTGACTCTTCTGCCCTTGAG4439                          ProSerAsnSerTyrProSerIleIleSerAspSerSerAlaLeuGlu                              1425143014351440                                                              GACCTGCGAAATCCAGAACAAAGCACATCAGAAAAAGCAGTATTAACT4487                          AspLeuArgAsnProGluGlnSerThrSerGluLysAlaValLeuThr                              144514501455                                                                  TCACAGAAAAGTAGTGAATACCCTATAAGCCAGAATCCAGAAGGCCTT4535                          SerGlnLysSerSerGluTyrProIleSerGlnAsnProGluGlyLeu                              146014651470                                                                  TCTGCTGACAAGTTTGAGGTGTCTGCAGATAGTTCTACCAGTAAAAAT4583                          SerAlaAspLysPheGluValSerAlaAspSerSerThrSerLysAsn                              147514801485                                                                  AAAGAACCAGGAGTGGAAAGGTCATCCCCTTCTAAATGCCCATCATTA4631                          LysGluProGlyValGluArgSerSerProSerLysCysProSerLeu                              149014951500                                                                  GATGATAGGTGGTACATGCACAGTTGCTCTGGGAGTCTTCAGAATAGA4679                          AspAspArgTrpTyrMetHisSerCysSerGlySerLeuGlnAsnArg                              1505151015151520                                                              AACTACCCATCTCAAGAGGAGCTCATTAAGGTTGTTGATGTGGAGGAG4727                          AsnTyrProSerGlnGluGluLeuIleLysValValAspValGluGlu                              152515301535                                                                  CAACAGCTGGAAGAGTCTGGGCCACACGATTTGACGGAAACATCTTAC4775                          GlnGlnLeuGluGluSerGlyProHisAspLeuThrGluThrSerTyr                              154015451550                                                                  TTGCCAAGGCAAGATCTAGAGGGAACCCCTTACCTGGAATCTGGAATC4823                          LeuProArgGlnAspLeuGluGlyThrProTyrLeuGluSerGlyIle                              155515601565                                                                  AGCCTCTTCTCTGATGACCCTGAATCTGATCCTTCTGAAGACAGAGCC4871                          SerLeuPheSerAspAspProGluSerAspProSerGluAspArgAla                              157015751580                                                                  CCAGAGTCAGCTCGTGTTGGCAACATACCATCTTCAACCTCTGCATTG4919                          ProGluSerAlaArgValGlyAsnIleProSerSerThrSerAlaLeu                              1585159015951600                                                              AAAGTTCCCCAATTGAAAGTTGCAGAATCTGCCCAGAGTCCAGCTGCT4967                          LysValProGlnLeuLysValAlaGluSerAlaGlnSerProAlaAla                              160516101615                                                                  GCTCATACTACTGATACTGCTGGGTATAATGCAATGGAAGAAAGTGTG5015                          AlaHisThrThrAspThrAlaGlyTyrAsnAlaMetGluGluSerVal                              162016251630                                                                  AGCAGGGAGAAGCCAGAATTGACAGCTTCAACAGAAAGGGTCAACAAA5063                          SerArgGluLysProGluLeuThrAlaSerThrGluArgValAsnLys                              163516401645                                                                  AGAATGTCCATGGTGGTGTCTGGCCTGACCCCAGAAGAATTTATGCTC5111                          ArgMetSerMetValValSerGlyLeuThrProGluGluPheMetLeu                              165016551660                                                                  GTGTACAAGTTTGCCAGAAAACACCACATCACTTTAACTAATCTAATT5159                          ValTyrLysPheAlaArgLysHisHisIleThrLeuThrAsnLeuIle                              1665167016751680                                                              ACTGAAGAGACTACTCATGTTGTTATGAAAACAGATGCTGAGTTTGTG5207                          ThrGluGluThrThrHisValValMetLysThrAspAlaGluPheVal                              168516901695                                                                  TGTGAACGGACACTGAAATATTTTCTAGGAATTGCGGGAGGAAAATGG5255                          CysGluArgThrLeuLysTyrPheLeuGlyIleAlaGlyGlyLysTrp                              170017051710                                                                  GTAGTTAGCTATTTCTGGGTGACCCAGTCTATTAAAGAAAGAAAAATG5303                          ValValSerTyrPheTrpValThrGlnSerIleLysGluArgLysMet                              171517201725                                                                  CTGAATGAGCATGATTTTGAAGTCAGAGGAGATGTGGTCAATGGAAGA5351                          LeuAsnGluHisAspPheGluValArgGlyAspValValAsnGlyArg                              173017351740                                                                  AACCACCAAGGTCCAAAGCGAGCAAGAGAATCCCAGGACAGAAAGATC5399                          AsnHisGlnGlyProLysArgAlaArgGluSerGlnAspArgLysIle                              1745175017551760                                                              TTCAGGGGGCTAGAAATCTGTTGCTATGGGCCCTTCACCAACATGCCC5447                          PheArgGlyLeuGluIleCysCysTyrGlyProPheThrAsnMetPro                              176517701775                                                                  ACAGATCAACTGGAATGGATGGTACAGCTGTGTGGTGCTTCTGTGGTG5495                          ThrAspGlnLeuGluTrpMetValGlnLeuCysGlyAlaSerValVal                              178017851790                                                                  AAGGAGCTTTCATCATTCACCCTTGGCACAGGTGTCCACCCAATTGTG5543                          LysGluLeuSerSerPheThrLeuGlyThrGlyValHisProIleVal                              179518001805                                                                  GTTGTGCAGCCAGATGCCTGGACAGAGGACAATGGCTTCCATGCAATT5591                          ValValGlnProAspAlaTrpThrGluAspAsnGlyPheHisAlaIle                              181018151820                                                                  GGGCAGATGTGTGAGGCACCTGTGGTGACCCGAGAGTGGGTGTTGGAC5639                          GlyGlnMetCysGluAlaProValValThrArgGluTrpValLeuAsp                              1825183018351840                                                              AGTGTAGCACTCTACCAGTGCCAGGAGCTGGACACCTACCTGATACCC5687                          SerValAlaLeuTyrGlnCysGlnGluLeuAspThrTyrLeuIlePro                              184518501855                                                                  CAGATCCCCCACAGCCACTACTGACTGCAGCCAGCCACAGGTACAGAGCCACAG5741                    GlnIleProHisSerHisTyr                                                         1860                                                                          GACCCCAAGAATGAGCTTACAAAGTGGCCTTTCCAGGCCCTGGGAGCTCCTCTCACTCTT5801              CAGTCCTTCTACTGTCCTGGCTACTAAATATTTTATGTACATCAGCCTGAAAAGGACTTC5861              TGGCTATGCAAGGGTCCCTTAAAGATTTTCTGCTTGAAGTCTCCCTTGGAAAT5914                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1863 amino acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAspLeuSerAlaLeuArgValGluGluValGlnAsnValIleAsn                              151015                                                                        AlaMetGlnLysIleLeuGluCysProIleCysLeuGluLeuIleLys                              202530                                                                        GluProValSerThrLysCysAspHisIlePheCysLysPheCysMet                              354045                                                                        LeuLysLeuLeuAsnGlnLysLysGlyProSerGlnCysProLeuCys                              505560                                                                        LysAsnAspIleThrLysArgSerLeuGlnGluSerThrArgPheSer                              65707580                                                                      GlnLeuValGluGluLeuLeuLysIleIleCysAlaPheGlnLeuAsp                              859095                                                                        ThrGlyLeuGluTyrAlaAsnSerTyrAsnPheAlaLysLysGluAsn                              100105110                                                                     AsnSerProGluHisLeuLysAspGluValSerIleIleGlnSerMet                              115120125                                                                     GlyTyrArgAsnArgAlaLysArgLeuLeuGlnSerGluProGluAsn                              130135140                                                                     ProSerLeuGlnGluThrSerLeuSerValGlnLeuSerAsnLeuGly                              145150155160                                                                  ThrValArgThrLeuArgThrLysGlnArgIleGlnProGlnLysThr                              165170175                                                                     SerValTyrIleGluLeuGlySerAspSerSerGluAspThrValAsn                              180185190                                                                     LysAlaThrTyrCysSerValGlyAspGlnGluLeuLeuGlnIleThr                              195200205                                                                     ProGlnGlyThrArgAspGluIleSerLeuAspSerAlaLysLysAla                              210215220                                                                     AlaCysGluPheSerGluThrAspValThrAsnThrGluHisHisGln                              225230235240                                                                  ProSerAsnAsnAspLeuAsnThrThrGluLysArgAlaAlaGluArg                              245250255                                                                     HisProGluLysTyrGlnGlySerSerValSerAsnLeuHisValGlu                              260265270                                                                     ProCysGlyThrAsnThrHisAlaSerSerLeuGlnHisGluAsnSer                              275280285                                                                     SerLeuLeuLeuThrLysAspArgMetAsnValGluLysAlaGluPhe                              290295300                                                                     CysAsnLysSerLysGlnProGlyLeuAlaArgSerGlnHisAsnArg                              305310315320                                                                  TrpAlaGlySerLysGluThrCysAsnAspArgArgThrProSerThr                              325330335                                                                     GluLysLysValAspLeuAsnAlaAspProLeuCysGluArgLysGlu                              340345350                                                                     TrpAsnLysGlnLysLeuProCysSerGluAsnProArgAspThrGlu                              355360365                                                                     AspValProTrpIleThrLeuAsnSerSerIleGlnLysValAsnGlu                              370375380                                                                     TrpPheSerArgSerAspGluLeuLeuGlySerAspAspSerHisAsp                              385390395400                                                                  GlyGluSerGluSerAsnAlaLysValAlaAspValLeuAspValLeu                              405410415                                                                     AsnGluValAspGluTyrSerGlySerSerGluLysIleAspLeuLeu                              420425430                                                                     AlaSerAspProHisGluAlaLeuIleCysLysSerGluArgValHis                              435440445                                                                     SerLysSerValGluSerAsnIleGluAspLysIlePheGlyLysThr                              450455460                                                                     TyrArgLysLysAlaSerLeuProAsnLeuSerHisValThrGluAsn                              465470475480                                                                  LeuIleIleGlyAlaPheValThrGluProGlnIleIleGlnGluArg                              485490495                                                                     ProLeuThrAsnLysLeuLysArgLysArgArgProThrSerGlyLeu                              500505510                                                                     HisProGluAspPheIleLysLysAlaAspLeuAlaValGlnLysThr                              515520525                                                                     ProGluMetIleAsnGlnGlyThrAsnGlnThrGluGlnAsnGlyGln                              530535540                                                                     ValMetAsnIleThrAsnSerGlyHisGluAsnLysThrLysGlyAsp                              545550555560                                                                  SerIleGlnAsnGluLysAsnProAsnProIleGluSerLeuGluLys                              565570575                                                                     GluSerAlaPheLysThrLysAlaGluProIleSerSerSerIleSer                              580585590                                                                     AsnMetGluLeuGluLeuAsnIleHisAsnSerLysAlaProLysLys                              595600605                                                                     AsnArgLeuArgArgLysSerSerThrArgHisIleHisAlaLeuGlu                              610615620                                                                     LeuValValSerArgAsnLeuSerProProAsnCysThrGluLeuGln                              625630635640                                                                  IleAspSerCysSerSerSerGluGluIleLysLysLysLysTyrAsn                              645650655                                                                     GlnMetProValArgHisSerArgAsnLeuGlnLeuMetGluGlyLys                              660665670                                                                     GluProAlaThrGlyAlaLysLysSerAsnLysProAsnGluGlnThr                              675680685                                                                     SerLysArgHisAspSerAspThrPheProGluLeuLysLeuThrAsn                              690695700                                                                     AlaProGlySerPheThrLysCysSerAsnThrSerGluLeuLysGlu                              705710715720                                                                  PheValAsnProSerLeuProArgGluGluLysGluGluLysLeuGlu                              725730735                                                                     ThrValLysValSerAsnAsnAlaGluAspProLysAspLeuMetLeu                              740745750                                                                     SerGlyGluArgValLeuGlnThrGluArgSerValGluSerSerSer                              755760765                                                                     IleSerLeuValProGlyThrAspTyrGlyThrGlnGluSerIleSer                              770775780                                                                     LeuLeuGluValSerThrLeuGlyLysAlaLysThrGluProAsnLys                              785790795800                                                                  CysValSerGlnCysAlaAlaPheGluAsnProLysGlyLeuIleHis                              805810815                                                                     GlyCysSerLysAspAsnArgAsnAspThrGluGlyPheLysTyrPro                              820825830                                                                     LeuGlyHisGluValAsnHisSerArgGluThrSerIleGluMetGlu                              835840845                                                                     GluSerGluLeuAspAlaGlnTyrLeuGlnAsnThrPheLysValSer                              850855860                                                                     LysArgGlnSerPheAlaProPheSerAsnProGlyAsnAlaGluGlu                              865870875880                                                                  GluCysAlaThrPheSerAlaHisSerGlySerLeuLysLysGlnSer                              885890895                                                                     ProLysValThrPheGluCysGluGlnLysGluGluAsnGlnGlyLys                              900905910                                                                     AsnGluSerAsnIleLysProValGlnThrValAsnIleThrAlaGly                              915920925                                                                     PheProValValGlyGlnLysAspLysProValAspAsnAlaLysCys                              930935940                                                                     SerIleLysGlyGlySerArgPheCysLeuSerSerGlnPheArgGly                              945950955960                                                                  AsnGluThrGlyLeuIleThrProAsnLysHisGlyLeuLeuGlnAsn                              965970975                                                                     ProTyrArgIleProProLeuPheProIleLysSerPheValLysThr                              980985990                                                                     LysCysLysLysAsnLeuLeuGluGluAsnPheGluGluHisSerMet                              99510001005                                                                   SerProGluArgGluMetGlyAsnGluAsnIleProSerThrValSer                              101010151020                                                                  ThrIleSerArgAsnAsnIleArgGluAsnValPheLysGluAlaSer                              1025103010351040                                                              SerSerAsnIleAsnGluValGlySerSerThrAsnGluValGlySer                              104510501055                                                                  SerIleAsnGluIleGlySerSerAspGluAsnIleGlnAlaGluLeu                              106010651070                                                                  GlyArgAsnArgGlyProLysLeuAsnAlaMetLeuArgLeuGlyVal                              107510801085                                                                  LeuGlnProGluValTyrLysGlnSerLeuProGlySerAsnCysLys                              109010951100                                                                  HisProGluIleLysLysGlnGluTyrGluGluValValGlnThrVal                              1105111011151120                                                              AsnThrAspPheSerProTyrLeuIleSerAspAsnLeuGluGlnPro                              112511301135                                                                  MetGlySerSerHisAlaSerGlnValCysSerGluThrProAspAsp                              114011451150                                                                  LeuLeuAspAspGlyGluIleLysGluAspThrSerPheAlaGluAsn                              115511601165                                                                  AspIleLysGluSerSerAlaValPheSerLysSerValGlnLysGly                              117011751180                                                                  GluLeuSerArgSerProSerProPheThrHisThrHisLeuAlaGln                              1185119011951200                                                              GlyTyrArgArgGlyAlaLysLysLeuGluSerSerGluGluAsnLeu                              120512101215                                                                  SerSerGluAspGluGluLeuProCysPheGlnHisLeuLeuPheGly                              122012251230                                                                  LysValAsnAsnIleProSerGlnSerThrArgHisSerThrValAla                              123512401245                                                                  ThrGluCysLeuSerLysAsnThrGluGluAsnLeuLeuSerLeuLys                              125012551260                                                                  AsnSerLeuAsnAspCysSerAsnGlnValIleLeuAlaLysAlaSer                              1265127012751280                                                              GlnGluHisHisLeuSerGluGluThrLysCysSerAlaSerLeuPhe                              128512901295                                                                  SerSerGlnCysSerGluLeuGluAspLeuThrAlaAsnThrAsnThr                              130013051310                                                                  GlnAspProPheLeuIleGlySerSerLysGlnMetArgHisGlnSer                              131513201325                                                                  GluSerGlnGlyValGlyLeuSerAspLysGluLeuValSerAspAsp                              133013351340                                                                  GluGluArgGlyThrGlyLeuGluGluAsnAsnGlnGluGluGlnSer                              1345135013551360                                                              MetAspSerAsnLeuGlyGluAlaAlaSerGlyCysGluSerGluThr                              136513701375                                                                  SerValSerGluAspCysSerGlyLeuSerSerGlnSerAspIleLeu                              138013851390                                                                  ThrThrGlnGlnArgAspThrMetGlnHisAsnLeuIleLysLeuGln                              139514001405                                                                  GlnGluMetAlaGluLeuGluAlaValLeuGluGlnHisGlySerGln                              141014151420                                                                  ProSerAsnSerTyrProSerIleIleSerAspSerSerAlaLeuGlu                              1425143014351440                                                              AspLeuArgAsnProGluGlnSerThrSerGluLysAlaValLeuThr                              144514501455                                                                  SerGlnLysSerSerGluTyrProIleSerGlnAsnProGluGlyLeu                              146014651470                                                                  SerAlaAspLysPheGluValSerAlaAspSerSerThrSerLysAsn                              147514801485                                                                  LysGluProGlyValGluArgSerSerProSerLysCysProSerLeu                              149014951500                                                                  AspAspArgTrpTyrMetHisSerCysSerGlySerLeuGlnAsnArg                              1505151015151520                                                              AsnTyrProSerGlnGluGluLeuIleLysValValAspValGluGlu                              152515301535                                                                  GlnGlnLeuGluGluSerGlyProHisAspLeuThrGluThrSerTyr                              154015451550                                                                  LeuProArgGlnAspLeuGluGlyThrProTyrLeuGluSerGlyIle                              155515601565                                                                  SerLeuPheSerAspAspProGluSerAspProSerGluAspArgAla                              157015751580                                                                  ProGluSerAlaArgValGlyAsnIleProSerSerThrSerAlaLeu                              1585159015951600                                                              LysValProGlnLeuLysValAlaGluSerAlaGlnSerProAlaAla                              160516101615                                                                  AlaHisThrThrAspThrAlaGlyTyrAsnAlaMetGluGluSerVal                              162016251630                                                                  SerArgGluLysProGluLeuThrAlaSerThrGluArgValAsnLys                              163516401645                                                                  ArgMetSerMetValValSerGlyLeuThrProGluGluPheMetLeu                              165016551660                                                                  ValTyrLysPheAlaArgLysHisHisIleThrLeuThrAsnLeuIle                              1665167016751680                                                              ThrGluGluThrThrHisValValMetLysThrAspAlaGluPheVal                              168516901695                                                                  CysGluArgThrLeuLysTyrPheLeuGlyIleAlaGlyGlyLysTrp                              170017051710                                                                  ValValSerTyrPheTrpValThrGlnSerIleLysGluArgLysMet                              171517201725                                                                  LeuAsnGluHisAspPheGluValArgGlyAspValValAsnGlyArg                              173017351740                                                                  AsnHisGlnGlyProLysArgAlaArgGluSerGlnAspArgLysIle                              1745175017551760                                                              PheArgGlyLeuGluIleCysCysTyrGlyProPheThrAsnMetPro                              176517701775                                                                  ThrAspGlnLeuGluTrpMetValGlnLeuCysGlyAlaSerValVal                              178017851790                                                                  LysGluLeuSerSerPheThrLeuGlyThrGlyValHisProIleVal                              179518001805                                                                  ValValGlnProAspAlaTrpThrGluAspAsnGlyPheHisAlaIle                              181018151820                                                                  GlyGlnMetCysGluAlaProValValThrArgGluTrpValLeuAsp                              1825183018351840                                                              SerValAlaLeuTyrGlnCysGlnGluLeuAspThrTyrLeuIlePro                              184518501855                                                                  GlnIleProHisSerHisTyr                                                         1860                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: s754 A                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CTAGCCTGGGCAACAAACGA20                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: s754 B                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCAGGAAGCAGGAATGGAAC20                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: s975 A                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TAGGAGATGGATTATTGGTG20                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: s975 B                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AGGCAACTTTGCAATGAGTG20                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: tdj1474 A                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CAGAGTGAGACCTTGTCTCAAA22                                                      (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: tdj1474 B                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TTCTGCAAACACCTTAAACTCAG23                                                     (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: tdj1239 A                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AACCTGGAAGGCAGAGGTTG20                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: tdj1239 B                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TCTGTACCTGCTAAGCAGTGG21                                                       (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 111 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2..111                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GGKCTTACTCTGTTGTCCCAGCTGGAGTACAGWGTGCGATCATGAG46                              XaaLeuLeuCysCysProSerTrpSerThrXaaCysAspHisGlu                                 186518701875                                                                  GCTTACTGTTGCTTGACTCCTAGGCTCAAGCGATCCTATCACCTCAGT94                            AlaTyrCysCysLeuThrProArgLeuLysArgSerTyrHisLeuSer                              1880188518901895                                                              CTCCAAGTAGCTGGACT111                                                          LeuGlnValAlaGly                                                               1900                                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      XaaLeuLeuCysCysProSerTrpSerThrXaaCysAspHisGluAla                              151015                                                                        TyrCysCysLeuThrProArgLeuLysArgSerTyrHisLeuSerLeu                              202530                                                                        GlnValAlaGly                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1534 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GAGGCTAGAGGGCAGGCACTTTATGGCAAACTCAGGTAGAATTCTTCCTCTTCCGTCTCT60                TTCCTTTTACGTCATCGGGGAGACTGGGTGGCAATCGCAGCCCGAGAGACGCATGGCTCT120               TTCTGCCCTCCATCCTCTGATGTACCTTGATTTCGTATTCTGAGAGGCTGCTGCTTAGCG180               GTAGCCCCTTGGTTTCCGTGGCAACGGAAAAGCGCGGGAATTACAGATAAATTAAAACTG240               CGACTGCGCGGCGTGAGCTCGCTGAGACTTCCTGGACCCCGCACCAGGCTGTGGGGTTTC300               TCAGATAACTGGGCCCCTGCGCTCAGGAGGCCTTCACCCTCTGCTCTGGGTAAAGGTAGT360               AGAGTCCCGGGAAAGGGACAGGGGGCCCAAGTGATGCTCTGGGGTACTGGCGTGGGAGAG420               TGGATTTCCGAAGCTGACAGATGGGTATTCTTTGACGGGGGGTAGGGGCGGAACCTGAGA480               GGCGTAAGGCGTTGTGAACCCTGGGGAGGGGGGCAGTTTGTAGGTCGCGAGGGAAGCGCT540               GAGGATCAGGAAGGGGGCACTGAGTGTCCGTGGGGGAATCCTCGTGATAGGAACTGGAAT600               ATGCCTTGAGGGGGACACTATGTCTTTAAAAACGTCGGCTGGTCATGAGGTCAGGAGTTC660               CAGACCAGCCTGACCAACGTGGTGAAACTCCGTCTCTACTAAAAATACNAAAATTAGCCG720               GGCGTGGTGCCGCTCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTAGAACCCGGGA780               GGCGGAGGTTGCAGTGAGCCGAGATCGCGCCATTGCACTCCAGCCTGGGCGACAGAGCGA840               GACTGTCTCAAAACAAAACAAAACAAAACAAAACAAAAAACACCGGCTGGTATGTATGAG900               AGGATGGGACCTTGTGGAAGAAGAGGTGCCAGGAATATGTCTGGGAAGGGGAGGAGACAG960               GATTTTGTGGGAGGGAGAACTTAAGAACTGGATCCATTTGCGCCATTGAGAAAGCGCAAG1020              AGGGAAGTAGAGGAGCGTCAGTAGTAACAGATGCTGCCGGCAGGGATGTGCTTGAGGAGG1080              ATCCAGAGATGAGAGCAGGTCACTGGGAAAGGTTAGGGGCGGGGAGGCCTTGATTGGTGT1140              TGGTTTGGTCGTTGTTGATTTTGGTTTTATGCAAGAAAAAGAAAACAACCAGAAACATTG1200              GAGAAAGCTAAGGCTACCACCACCTACCCGGTCAGTCACTCCTCTGTAGCTTTCTCTTTC1260              TTGGAGAAAGGAAAAGACCCAAGGGGTTGGCAGCGATATGTGAAAAAATTCAGAATTTAT1320              GTTGTCTAATTACAAAAAGCAACTTCTAGAATCTTTAAAAATAAAGGACGTTGTCATTAG1380              TTCTTCTGGTTTGTATTATTCTAAAACCTTCCAAATCTTCAAATTTACTTTATTTTAAAA1440              TGATAAAATGAAGTTGTCATTTTATAAACCTTTTAAAAAGATATATATATATGTTTTTCT1500              AATGTGTTAAAGTTCATTGGAACAGAAAGAAATG1534                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1924 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GAGGCTAGAGGGCAGGCACTTTATGGCAAACTCAGGTAGAATTCTTCCTCTTCCGTCTCT60                TTCCTTTTACGTCATCGGGGAGACTGGGTGGCAATCGCAGCCCGAGAGACGCATGGCTCT120               TTCTGCCCTCCATCCTCTGATGTACCTTGATTTCGTATTCTGAGAGGCTGCTGCTTAGCG180               GTAGCCCCTTGGTTTCCGTGGCAACGGAAAAGCGCGGGAATTACAGATAAATTAAAACTG240               CGACTGCGCGGCGTGAGCTCGCTGAGACTTCCTGGACCCCGCACCAGGCTGTGGGGTTTC300               TCAGATAACTGGGCCCCTGCGCTCAGGAGGCCTTCACCCTCTGCTCTGGGTAAAGGTAGT360               AGAGTCCCGGGAAAGGGACAGGGGGCCCAAGTGATGCTCTGGGGTACTGGCGTGGGAGAG420               TGGATTTCCGAAGCTGACAGATGGGTATTCTTTGACGGGGGGTAGGGGCGGAACCTGAGA480               GGCGTAAGGCGTTGTGAACCCTGGGGAGGGGGGCAGTTTGTAGGTCGCGAGGGAAGCGCT540               GAGGATCAGGAAGGGGGCACTGAGTGTCCGTGGGGGAATCCTCGTGATAGGAACTGGAAT600               ATGCCTTGAGGGGGACACTATGTCTTTAAAAACGTCGGCTGGTCATGAGGTCAGGAGTTC660               CAGACCAGCCTGACCAACGTGGTGAAACTCCGTCTCTACTAAAAATACNAAAATTAGCCG720               GGCGTGGTGCCGCTCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTAGAACCCGGGA780               GGCGGAGGTTGCAGTGAGCCGAGATCGCGCCATTGCACTCCAGCCTGGGCGACAGAGCGA840               GACTGTCTCAAAACAAAACAAAACAAAACAAAACAAAAAACACCGGCTGGTATGTATGAG900               AGGATGGGACCTTGTGGAAGAAGAGGTGCCAGGAATATGTCTGGGAAGGGGAGGAGACAG960               GATTTTGTGGGAGGGAGAACTTAAGAACTGGATCCATTTGCGCCATTGAGAAAGCGCAAG1020              AGGGAAGTAGAGGAGCGTCAGTAGTAACAGATGCTGCCGGCAGGGATGTGCTTGAGGAGG1080              ATCCAGAGATGAGAGCAGGTCACTGGGAAAGGTTAGGGGCGGGGAGGCCTTGATTGGTGT1140              TGGTTTGGTCGTTGTTGATTTTGGTTTTATGCAAGAAAAAGAAAACAACCAGAAACATTG1200              GAGAAAGCTAAGGCTACCACCACCTACCCGGTCAGTCACTCCTCTGTAGCTTTCTCTTTC1260              TTGGAGAAAGGAAAAGACCCAAGGGGTTGGCAGCGATATGTGAAAAAATTCAGAATTTAT1320              GTTGTCTAATTACAAAAAGCAACTTCTAGAATCTTTAAAAATAAAGGACGTTGTCATTAG1380              TTCTTCTGGTTTGTATTATTCTAAAACCTTCCAAATCTTCAAATTTACTTTATTTTAAAA1440              TGATAAAATGAAGTTGTCATTTTATAAACCTTTTAAAAAGATATATATATATGTTTTTCT1500              AATGTGTTAAAGTTCATTGGAACAGAAAGAAATGGATTTATCTGCTCTTCGCGTTGAAGA1560              AGTACAAAATGTCATTAATGCTATGCAGAAAATCTTAGAGTGTCCCATCTGGTAAGTCAG1620              CACAAGAGTGTATTAATTTGGGATTCCTATGATTATCTCCTATGCAAATGAACAGAATTG1680              ACCTTACATACTAGGGAAGAAAAGACATGTCTAGTAAGATTAGGCTATTGTAATTGCTGA1740              TTTTCTTAACTGAAGAACTTTAAAAATATAGAAAATGATTCCTTGTTCTCCATCCACTCT1800              GCCTCTCCCACTCCTCTCCTTTTCAACACAATCCTGTGGTCCGGGAAAGACAGGGCTCTG1860              TCTTGATTGGTTCTGCACTGGGCAGGATCTGTTAGATACTGCATTTGCTTTCTCCAGCTC1920              TAAA1924                                                                      (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 631 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AAATGCTGATGATAGTATAGAGTATTGAAGGGATCAATATAATTCTGTTTTGATATCTGA60                AAGCTCACTGAAGGTAAGGATCGTATTCTCTGCTGTATTCTCAGTTCCTGACACAGCAGA120               CATTTAATAAATATTGAACGAACTTGAGGCCTTATGTTGACTCAGTCATAACAGCTCAAA180               GTTGAACTTATTCACTAAGAATAGCTTTATTTTTAAATAAATTATTGAGCCTCATTTATT240               TTCTTTTTCTCCCCCCCCTACCCTGCTAGTCTGGAGTTGATCAAGGAACCTGTCTCCACA300               AAGTGTGACCACATATTTTGCAAGTAAGTTTGAATGTGTTATGTGGCTCCATTATTAGCT360               TTTGTTTTTGTCCTTCATAACCCAGGAAACACCTAACTTTATAGAAGCTTTACTTTCTTC420               AATTAAGTGAGAACGAAAATCCAACTCCATTTCATTCTTTCTCAGAGAGTATATAGTTAT480               CAAAAGTTGGTTGTAATCATAGTTCCTGGTAAAGTTTTGACATATATTATCTTTTTTTTT540               TTTTGAGACAAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCATGAGGCTTGCTCA600               CTGCACCTCCGCCCCCGAGTTCAGCGACTCT631                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 481 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      TGAGATCTAGACCACATGGTCAAAGAGATAGAATGTGAGCAATAAATGAACCTTAAATTT60                TTCAACAGCTACTTTTTTTTTTTTTTTTTGAGACAGGGKCTTACTCTGTTGTCCCAGCTG120               GAGTACAGWGTGCGATCATGAGGCTTACTGTTGCTTGACTCCTAGGCTCAAGCGATCCTA180               TCACCTCAGTCTCCAAGTAGCTGGACTGTAAGTGCACACCACCATATCCAGCTAAATTTT240               GTGTTTTCTGTAGAGACGGGGTTTCGCCATGTTTCCCAGGCTGGTCTTGAACTTTGGGCT300               TAACCCGTCTGCCCACCTAGGCATCCCAAAGTGCTAGGATTACAGGTGTGAGTCATCATG360               CCTGGCCAGTATTTTAGTTAGCTCTGTCTTTTCAAGTCATATACAAGTTCATTTTCTTTT420               AAGTTTAGTTAACAACCTTATATCATGTATTCTTTTCTAGCATAAAGAAAGATTCGAGGC480               C481                                                                          (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 522 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TGTGATCATAACAGTAAGCCATATGCATGTAAGTTCAGTTTTCATAGATCATTGCTTATG60                TAGTTTAGGTTTTTGCTTATGCAGCATCCAAAAACAATTAGGAAACTATTGCTTGTAATT120               CACCTGCCATTACTTTTTAAATGGCTCTTAAGGGCAGTTGTGAGATTATCTTTTCATGGC180               TATTTGCCTTTTGAGTATTCTTTCTACAAAAGGAAGTAAATTAAATTGTTCTTTCTTTCT240               TTATAATTTATAGATTTTGCATGCTGAAACTTCTCAACCAGAAGAAAGGGCCTTCACAGT300               GTCCTTTATGTAAGAATGATATAACCAAAAGGTATATAATTTGGTAATGATGCTAGGTTG360               GAAGCAACCACAGTAGGAAAAAGTAGAAATTATTTAATAACATAGCGTTCCTATAAAACC420               ATTCATCAGAAAAATTTATAAAAGAGTTTTTAGCACACAGTAAATTATTTCCAAAGTTAT480               TTTCCTGAAAGTTTTATGGGCATCTGCCTTATACAGGTATTG522                                 (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 465 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GGTAGGCTTAAATGAATGACAAAAAGTTACTAAATCACTGCCATCACACGGTTTATACAG60                ATGTCAATGATGTATTGATTATAGAGGTTTTCTACTGTTGCTGCATCTTATTTTTATTTG120               TTTACATGTCTTTTCTTATTTTAGTGTCCTTAAAAGGTTGATAATCACTTGCTGAGTGTG180               TTTCTCAAACAATTTAATTTCAGGAGCCTACAAGAAAGTACGAGATTTAGTCAACTTGTT240               GAAGAGCTATTGAAAATCATTTGTGCTTTTCAGCTTGACACAGGTTTGGAGTGTAAGTGT300               TGAATATCCCAAGAATGACACTCAAGTGCTGTCCATGAAAACTCAGGAAGTTTGCACAAT360               TACTTTCTATGACGTGGTGATAAGACCTTTTAGTCTAGGTTAATTTTAGTTCTGTATCTG420               TAATCTATTTTAAAAAATTACTCCCACTGGTCTCACACCTTATTT465                              (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 513 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AAAAAATCACAGGTAACCTTAATGCATTGTCTTAACACAACAAAGAGCATACATAGGGTT60                TCTCTTGGTTTCTTTGATTATAATTCATACATTTTTCTCTAACTGCAAACATAATGTTTT120               CCCTTGTATTTTACAGATGCAAACAGCTATAATTTTGCAAAAAAGGAAAATAACTCTCCT180               GAACATCTAAAAGATGAAGTTTCTATCATCCAAAGTATGGGCTACAGAAACCGTGCCAAA240               AGACTTCTACAGAGTGAACCCGAAAATCCTTCCTTGGTAAAACCATTTGTTTTCTTCTTC300               TTCTTCTTCTTCTTTTCTTTTTTTTTTCTTTTTTTTTTTGAGATGGAGTCTTGCTCTGTG360               GCCCAGGCTAGAAGCAGTCCTCCTGCCTTAGCCNCCTTAGTAGCTGGGATTACAGGCACG420               CGCACCATGCCAGGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCATCATGTTGGCC480               AGGCTGGTCTCGAACTCCTAACCTCAGGTGATC513                                          (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6769 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      ATGATGGAGATCTTAAAAAGTAATCATTCTGGGGCTGGGCGTAGTAGCTTGCACCTGTAA60                TCCCAGCACTTCGGGAGGCTGAGGCAGGCAGATAATTTGAGGTCAGGAGTTTGAGACCAG120               CCTGGCCAACATGGTGAAACCCATCTCTACTAAAAATACAAAAATTAGCTGGGTGTGGTG180               GCACGTACCTGTAATCCCAGCTACTCGGGAGGCGGAGGCACAAGAATTGCTTGAACCTAG240               GACGCGGAGGTTGCAGCGAGCCAAGATCGCGCCACTGCACTCCAGCCTGGGCCGTAGAGT300               GAGACTCTGTCTCAAAAAAGAAAAAAAAGTAATTGTTCTAGCTGGGCGCAGTGGCTCTTG360               CCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGTGGATCTCGAGTCCTAGAGTTCAAG420               ACCAGCCTAGGCAATGTGGTGAAACCCCATCGCTACAAAAAATACAAAAATTAGCCAGGC480               ATGGTGGCGTGCGCATGTAGTCCCAGCTCCTTGGGAGGCTGAGGTGGGAGGATCACTTGA540               ACCCAGGAGACAGAGGTTGCAGTGAACCGAGATCACGCCACCACGCTCCAGCCTGGGCAA600               CAGAACAAGACTCTGTCTAAAAAAATACAAATAAAATAAAAGTAGTTCTCACAGTACCAG660               CATTCATTTTTCAAAAGATATAGAGCTAAAAAGGAAGGAAAAAAAAAGTAATGTTGGGCT720               TTTAAATACTCGTTCCTATACTAAATGTTCTTAGGAGTGCTGGGGTTTTATTGTCATCAT780               TTATCCTTTTTAAAAATGTTATTGGCCAGGCACGGTGGCTCATGGCTGTAATCCCAGCAC840               TTTGGGAGGCCGAGGCAGGCAGATCACCTGAGGTCAGGAGTGTGAGACCAGCCTGGCCAA900               CATGGCGAAACCTGTCTCTACTAAAAATACAAAAATTAACTAGGCGTGGTGGTGTACGCC960               TGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCAACTGAACCAGGGAGGTGGAG1020              GTTGCAGTGTGCCGAGATCACGCCACTGCACTCTAGCCTGGCAACAGAGCAAGATTCTGT1080              CTCAAAAAAAAAAAACATATATACACATATATCCCAAAGTGCTGGGATTACATATATATA1140              TATATATATATATTATATATATATATATATATATATGTGATATATATGTGATATATATAT1200              AACATATATATATGTAATATATATGTGATATATATATAATATATATATGTAATATATATG1260              TGATATATATATATACACACACACACACATATATATGTATGTGTGTGTACACACACACAC1320              ACAAATTAGCCAGGCATAGTTGCACACGCTTGGTAGACCCAGCTACTCAGGAGGCTGAGG1380              GAGGAGAATCTCTTGAACTTAGGAGGCGGAGGTTGCAGTGAGCTGAGATTGCGCCACTGC1440              ACTCCAGCCTGGGTGACAGAGCAGGACTCTGTACACCCCCCAAAACAAAAAAAAAAGTTA1500              TCAGATGTGATTGGAATGTATATCAAGTATCAGCTTCAAAATATGCTATATTAATACTTC1560              AAAAATTACACAAATAATACATAATCAGGTTTGAAAAATTTAAGACAACMSAARAAAAAA1620              WYCMAATCACAMATATCCCACACATTTTATTATTMCTMCTMCWATTATTTTGWAGAGMCT1680              GGGTCTCACYCYKTTGCTWATGCTGGTCTTTGAACYCCYKGCCYCAARCARTCCTSCTCC1740              ABCCTCCCAARGTGCTGGGGATWATAGGCATGARCTAACCGCACCCAGCCCCAGACATTT1800              TAGTGTGTAAATTCCTGGGCATTTTTTCAAGGCATCATACATGTTAGCTGACTGATGATG1860              GTCAATTTATTTTGTCCATGGTGTCAAGTTTCTCTTCAGGAGGAAAAGCACAGAACTGGC1920              CAACAATTGCTTGACTGTTCTTTACCATACTGTTTAGCAGGAAACCAGTCTCAGTGTCCA1980              ACTCTCTAACCTTGGAACTGTGAGAACTCTGAGGACAAAGCAGCGGATACAACCTCAAAA2040              GACGTCTGTCTACATTGAATTGGGTAAGGGTCTCAGGTTTTTTAAGTATTTAATAATAAT2100              TGCTGGATTCCTTATCTTATAGTTTTGCCAAAAATCTTGGTCATAATTTGTATTTGTGGT2160              AGGCAGCTTTGGGAAGTGAATTTTATGAGCCCTATGGTGAGTTATAAAAAATGTAAAAGA2220              CGCAGTTCCCACCTTGAAGAATCTTACTTTAAAAAGGGAGCAAAAGAGGCCAGGCATGGT2280              GGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCAAAGTGGGTGGATCACCTGAGGTCG2340              GGAGTTCGAGACCAGCCTAGCCAACATGGAGAAACTCTGTCTGTACCAAAAAATAAAAAA2400              TTAGCCAGGTGTGGTGGCACATAACTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAG2460              AATCACTTGAACCCGGGAGGTGGAGGTTGCGGTGAACCGAGATCGCACCATTGCACTCCA2520              GCCTGGGCAAAAATAGCGAAACTCCATCTAAAAAAAAAAAAGAGAGCAAAAGAAAGAMTM2580              TCTGGTTTTAAMTMTGTGTAAATATGTTTTTGGAAAGATGGAGAGTAGCAATAAGAAAAA2640              ACATGATGGATTGCTACAGTATTTAGTTCCAAGATAAATTGTACTAGATGAGGAAGCCTT2700              TTAAGAAGAGCTGAATTGCCAGGCGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGA2760              GGCCGAGGTGGGCGGATCACCTGAGGTCGGGAGTTCAAGACCAGCCTGACCAACATGGAG2820              AAACCCCATCTCTACTAAAAAAAAAAAAAAAAAAATTAGCCGGGGTGGTGGCTTATGCCT2880              GTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTTGAACCCAGGAAGCAGAGG2940              TTGCAGTGAGCCAAGATCGCACCATTGCACTCCAGCCTAGGCAACAAGAGTGAAACTCCA3000              TCTCAAAAAAAAAAAAAAAGAGCTGAATCTTGGCTGGGCAGGATGGCTCGTGCCTGTAAT3060              CCTAACGCTTTGGAAGACCGAGGCAGAAGGATTGGTTGAGTCCACGAGTTTAAGACCAGC3120              CTGGCCAACATAGGGGAACCCTGTCTCTATTTTTAAAATAATAATACATTTTTGGCCGGT3180              GCGGTGGCTCATGCCTGTAATCCCAATACTTTGGGAGGCTGAGGCAGGTAGATCACCTGA3240              GGTCAGAGTTCGAGACCAGCCTGGATAACCTGGTGAAACCCCTCTTTACTAAAAATACAA3300              AAAAAAAAAAAAATTAGCTGGGTGTGGTAGCACATGCTTGTAATCCCAGCTACTTGGGAG3360              GCTGAGGCAGGAGAATCGCTTGAACCAGGGAGGCGGAGGTTACAATGAGCCAACACTACA3420              CCACTGCACTCCAGCCTGGGCAATAGAGTGAGACTGCATCTCAAAAAAATAATAATTTTT3480              AAAAATAATAAATTTTTTTAAGCTTATAAAAAGAAAAGTTGAGGCCAGCATAGTAGCTCA3540              CATCTGTAATCTCAGCAGTGGCAGAGGATTGCTTGAAGCCAGGAGTTTGAGACCAGCCTG3600              GGCAACATAGCAAGACCTCATCTCTACAAAAAAATTTCTTTTTTAAATTAGCTGGGTGTG3660              GTGGTGTGCATCTGTAGTCCCAGCTACTCAGGAGGCAGAGGTGAGTGGATACATTGAACC3720              CAGGAGTTTGAGGCTGTAGTGAGCTATGATCATGCCACTGCACTCCAACCTGGGTGACAG3780              AGCAAGACCTCCAAAAAAAAAAAAAAAAGAGCTGCTGAGCTCAGAATTCAAACTGGGCTC3840              TCAAATTGGATTTTCTTTTAGAATATATTTATAATTAAAAAGGATAGCCATCTTTTGAGC3900              TCCCAGGCACCACCATCTATTTATCATAACACTTACTGTTTTCCCCCCTTATGATCATAA3960              ATTCCTAGACAACAGGCATTGTAAAAATAGTTATAGTAGTTGATATTTAGGAGCACTTAA4020              CTATATTCCAGGCACTATTGTGCTTTTCTTGTATAACTCATTAGATGCTTGTCAGACCTC4080              TGAGATTGTTCCTATTATACTTATTTTACAGATGAGAAAATTAAGGCACAGAGAAGTTAT4140              GAAATTTTTCCAAGGTATTAAACCTAGTAAGTGGCTGAGCCATGATTCAAACCTAGGAAG4200              TTAGATGTCAGAGCCTGTGCTTTTTTTTTGTTTTTGTTTTTGTTTTCAGTAGAAACGGGG4260              GTCTCACTTTGTTGGCCAGGCTGGTCTTGAACTCCTAACCTCAAATAATCCACCCATCTC4320              GGCCTCCTCAAGTGCTGGGATTACAGGTGAGAGCCACTGTGCCTGGCGAAGCCCATGCCT4380              TTAACCACTTCTCTGTATTACATACTAGCTTAACTAGCATTGTACCTGCCACAGTAGATG4440              CTCAGTAAATATTTCTAGTTGAATATCTGTTTTTCAACAAGTACATTTTTTTAACCCTTT4500              TAATTAAGAAAACTTTTATTGATTTATTTTTTGGGGGGAAATTTTTTAGGATCTGATTCT4560              TCTGAAGATACCGTTAATAAGGCAACTTATTGCAGGTGAGTCAAAGAGAACCTTTGTCTA4620              TGAAGCTGGTATTTTCCTATTTAGTTAATATTAAGGATTGATGTTTCTCTCTTTTTAAAA4680              ATATTTTAACTTTTATTTTAGGTTCAGGGATGTATGTGCAGTTTGTTATATAGGTAAACA4740              CACGACTTGGGATTTGGTGTATAGATTTTTTTCATCATCCGGGTACTAAGCATACCCCAC4800              AGTTTTTTGTTTGCTTTCTTTCTGAATTTCTCCCTCTTCCCACCTTCCTCCCTCAAGTAG4860              GCTGGTGTTTCTCCAGACTAGAATCATGGTATTGGAAGAAACCTTAGAGATCATCTAGTT4920              TAGTTCTCTCATTTTATAGTGGAGGAAATACCCTTTTTGTTTGTTGGATTTAGTTATTAG4980              CACTGTCCAAAGGAATTTAGGATAACAGTAGAACTCTGCACATGCTTGCTTCTAGCAGAT5040              TGTTCTCTAAGTTCCTCATATACAGTAATATTGACACAGCAGTAATTGTGACTGATGAAA5100              ATGTTCAAGGACTTCATTTTCAACTCTTTCTTTCCTCTGTTCCTTATTTCCACATATCTC5160              TCAAGCTTTGTCTGTATGTTATATAATAAACTACAAGCAACCCCAACTATGTTACCTACC5220              TTCCTTAGGAATTATTGCTTGACCCAGGTTTTTTTTTTTTTTTTTTTGGAGACGGGGTCT5280              TGCCCTGTTGCCAGGATGGAGTGTAGTGGCGCCATCTCGGCTCACTGCAATCTCCAACTC5340              CCTGGTTCAAGCGATTCTCCTGTCTCAATCTCACGAGTAGCTGGGACTACAGGTATACAC5400              CACCACGCCCGGTTAATTGACCATTCCATTTCTTTCTTTCTCTCTTTTTTTTTTTTTTTT5460              TTGAGACAGAGTCTTGCTCTGTTGCCCAGGCTGGAGTACAGAGGTGTGATCTCACCTCTC5520              CGCAACGTCTGCCTCCCAGGTTGAAGCCATACTCCTGCCTCAGCCTCTCTAGTAGCTGGG5580              ACTACAGGCGCGCGCCACCACACCCGGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTT5640              CACCATGTTGGCCAGGCTGGTCTTGAACTCATGACCTCAAGTGGTCCACCCGCCTCAGCC5700              TCCCAAAGTGCTGGAATTACAGGCTTGAGCCACCGTGCCCAGCAACCATTTCATTTCAAC5760              TAGAAGTTTCTAAAGGAGAGAGCAGCTTTCACTAACTAAATAAGATTGGTCAGCTTTCTG5820              TAATCGAAAGAGCTAAAATGTTTGATCTTGGTCATTTGACAGTTCTGCATACATGTAACT5880              AGTGTTTCTTATTAGGACTCTGTCTTTTCCCTATAGTGTGGGAGATCAAGAATTGTTACA5940              AATCACCCCTCAAGGAACCAGGGATGAAATCAGTTTGGATTCTGCAAAAAAGGGTAATGG6000              CAAAGTTTGCCAACTTAACAGGCACTGAAAAGAGAGTGGGTAGATACAGTACTGTAATTA6060              GATTATTCTGAAGACCATTTGGGACCTTTACAACCCACAAAATCTCTTGGCAGAGTTAGA6120              GTATCATTCTCTGTCAAATGTCGTGGTATGGTCTGATAGATTTAAATGGTACTAGACTAA6180              TGTACCTATAATAAGACCTTCTTGTAACTGATTGTTGCCCTTTCGCTTTTTTTTTTGTTT6240              GTTTGTTTGTTTTTTTTTGAGATGGGGTCTCACTCTGTTGCCCAGGCTGGAGTGCAGTGA6300              TGCAATCTTGGCTCACTGCAACCTCCACCTCCAAAGGCTCAAGCTATCCTCCCACTTCAG6360              CCTCCTGAGTAGCTGGGACTACAGGCGCATGCCACCACACCCGGTTAATTTTTTGTGGTT6420              TTATAGAGATGGGGTTTCACCATGTTACCGAGGCTGGTCTCAAACTCCTGGACTCAAGCA6480              GTCTGCCCACTTCAGCCTCCCAAAGTGCTGCAGTTACAGGCTTGAGCCACTGTGCCTGGC6540              CTGCCCTTTACTTTTAATTGGTGTATTTGTGTTTCATCTTTTACCTACTGGTTTTTAAAT6600              ATAGGGAGTGGTAAGTCTGTAGATAGAACAGAGTATTAAGTAGACTTAATGGCCAGTAAT6660              CTTTAGAGTACATCAGAACCAGTTTTCTGATGGCCAATCTGCTTTTAATTCACTCTTAGA6720              CGTTAGAGAAATAGGTGTGGTTTCTGCATAGGGAAAATTCTGAAATTAA6769                         (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4249 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GATCCTAAGTGGAAATAATCTAGGTAAATAGGAATTAAATGAAAGAGTATGAGCTACATC60                TTCAGTATACTTGGTAGTTTATGAGGTTAGTTTCTCTAATATAGCCAGTTGGTTGATTTC120               CACCTCCAAGGTGTATGAAGTATGTATTTTTTTAATGACAATTCAGTTTTTGAGTACCTT180               GTTATTTTTGTATATTTTCAGCTGCTTGTGAATTTTCTGAGACGGATGTAACAAATACTG240               AACATCATCAACCCAGTAATAATGATTTGAACACCACTGAGAAGCGTGCAGCTGAGAGGC300               ATCCAGAAAAGTATCAGGGTAGTTCTGTTTCAAACTTGCATGTGGAGCCATGTGGCACAA360               ATACTCATGCCAGCTCATTACAGCATGAGAACAGCAGTTTATTACTCACTAAAGACAGAA420               TGAATGTAGAAAAGGCTGAATTCTGTAATAAAAGCAAACAGCCTGGCTTAGCAAGGAGCC480               AACATAACAGATGGGCTGGAAGTAAGGAAACATGTAATGATAGGCGGACTCCCAGCACAG540               AAAAAAAGGTAGATCTGAATGCTGATCCCCTGTGTGAGAGAAAAGAATGGAATAAGCAGA600               AACTGCCATGCTCAGAGAATCCTAGAGATACTGAAGATGTTCCTTGGATAACACTAAATA660               GCAGCATTCAGAAAGTTAATGAGTGGTTTTCCAGAAGTGATGAACTGTTAGGTTCTGATG720               ACTCACATGATGGGGAGTCTGAATCAAATGCCAAAGTAGCTGATGTATTGGACGTTCTAA780               ATGAGGTAGATGAATATTCTGGTTCTTCAGAGAAAATAGACTTACTGGCCAGTGATCCTC840               ATGAGGCTTTAATATGTAAAAGTGAAAGAGTTCACTCCAAATCAGTAGAGAGTAATATTG900               AAGGCCAAATATTTGGGAAAACCTATCGGAAGAAGGCAAGCCTCCCCAACTTAAGCCATG960               TAACTGAAAATCTAATTATAGGAGCATTTGTTACTGAGCCACAGATAATACAAGAGCGTC1020              CCCTCACAAATAAATTAAAGCGTAAAAGGAGACCTACATCAGGCCTTCATCCTGAGGATT1080              TTATCAAGAAAGCAGATTTGGCAGTTCAAAAGACTCCTGAAATGATAAATCAGGGAACTA1140              ACCAAACGGAGCAGAATGGTCAAGTGATGAATATTACTAATAGTGGTCATGAGAATAAAA1200              CAAAAGGTGATTCTATTCAGAATGAGAAAAATCCTAACCCAATAGAATCACTCGAAAAAG1260              AATCTGCTTTCAAAACGAAAGCTGAACCTATAAGCAGCAGTATAAGCAATATGGAACTCG1320              AATTAAATATCCACAATTCAAAAGCACCTAAAAAGAATAGGCTGAGGAGGAAGTCTTCTA1380              CCAGGCATATTCATGCGCTTGAACTAGTAGTCAGTAGAAATCTAAGCCCACCTAATTGTA1440              CTGAATTGCAAATTGATAGTTGTTCTAGCAGTGAAGAGATAAAGAAAAAAAAGTACAACC1500              AAATGCCAGTCAGGCACAGCAGAAACCTACAACTCATGGAAGGTAAAGAACCTGCAACTG1560              GAGCCAAGAAGAGTAACAAGCCAAATGAACAGACAAGTAAAAGACATGACAGCGATACTT1620              TCCCAGAGCTGAAGTTAACAAATGCACCTGGTTCTTTTACTAAGTGTTCAAATACCAGTG1680              AACTTAAAGAATTTGTCAATCCTAGCCTTCCAAGAGAAGAAAAAGAAGAGAACTAGAAAC1740              AGTTAAAGTGTCTAATAATGCTGAAGACCCCAAAGATCTCATGTTAAGTGGAGAAAGGGT1800              TTTGCAAACTGAAAGATCTGTAGAGAGTAGCAGTATTTCATTGGTACCTGGTACTGATTA1860              TGGCACTCAGGAAAGTATCTCGTTACTGGAAGTTAGCACTCTAGGGAAGGCAAAAACAGA1920              ACCAAATAAATGTGTGAGTCAGTGTGCAGCATTTGAAAACCCCAAGGGACTAATTCATGG1980              TTGTTCCAAAGATAATAGAAATGACACAGAAGGCTTTAAGTATCCATTGGGACATGAAGT2040              TAACCACAGTCGGGAAACAAGCATAGAAATGGAAGAAAGTGAACTTGATGCTCAGTATTT2100              GCAGAATACATTCAAGGTTTCAAAGCGCCAGTCATTTGCTCCGTTTTCAAATCCAGGAAA2160              TGCAGAAGAGGAATGTGCAACATTCTCTGCCCACTCTGGGTCCTTAAAGAAACAAAGTCC2220              AAAAGTCACTTTTGAATGTGAACAAAAGGAAGAAAATCAAGGAAAGAATGAGTCTAATAT2280              CAAGCCTGTACAGACAGTTAATATCACTGCAGGCTTTCCTGTGGTTGGTCAGAAAGATAA2340              GCCAGTTGATAATGCCAAATGTAGTATCAAAGGAGGCTCTAGGTTTTGTCTATCATCTCA2400              GTTCAGAGGCAACGAAACTGGACTCATTACTCCAAATAAACATGGACTTTTACAAAACCC2460              ATATCGTATACCACCACTTTTTCCCATCAAGTCATTTGTTAAAACTAAATGTAAGAAAAA2520              TCTGCTAGAGGAAAACTTTGAGGAACATTCAATGTCACCTGAAAGAGAAATGGGAAATGA2580              GAACATTCCAAGTACAGTGAGCACAATTAGCCGTAATAACATTAGAGAAAATGTTTTTAA2640              AGAAGCCAGCTCAAGCAATATTAATGAAGTAGGTTCCAGTACTAATGAAGTGGGCTCCAG2700              TATTAATGAAATAGGTTCCAGTGATGAAAACATTCAAGCAGAACTAGGTAGAAACAGAGG2760              GCCAAAATTGAATGCTATGCTTAGATTAGGGGTTTTGCAACCTGAGGTCTATAAACAAAG2820              TCTTCCTGGAAGTAATTGTAAGCATCCTGAAATAAAAAAGCAAGAATATGAAGAAGTAGT2880              TCAGACTGTTAATACAGATTTCTCTCCATATCTGATTTCAGATAACTTAGAACAGCCTAT2940              GGGAAGTAGTCATGCATCTCAGGTTTGTTCTGAGACACCTGATGACCTGTTAGATGATGG3000              TGAAATAAAGGAAGATACTAGTTTTGCTGAAAATGACATTAAGGAAAGTTCTGCTGTTTT3060              TAGCAAAAGCGTCCAGAAAGGAGAGCTTAGCAGGAGTCCTAGCCCTTTCACCCATACACA3120              TTTGGCTCAGGGTTACCGAAGAGGGGCCAAGAAATTAGAGTCCTCAGAAGAGAACTTATC3180              TAGTGAGGATGAAGAGCTTCCCTGCTTCCAACACTTGTTATTTGGTAAAGTAAACAATAT3240              ACCTTCTCAGTCTACTAGGCATAGCACCGTTGCTACCGAGTGTCTGTCTAAGAACACAGA3300              GGAGAATTTATTATCATTGAAGAATAGCTTAAATGACTGCAGTAACCAGGTAATATTGGC3360              AAAGGCATCTCAGGAACATCACCTTAGTGAGGAAACAAAATGTTCTGCTAGCTTGTTTTC3420              TTCACAGTGCAGTGAATTGGAAGACTTGACTGCAAATACAAACACCCAGGATCCTTTCTT3480              GATTGGTTCTTCCAAACAAATGAGGCATCAGTCTGAAAGCCAGGGAGTTGGTCTGAGTGA3540              CAAGGAATTGGTTTCAGATGATGAAGAAAGAGGAACGGGCTTGGAAGAAAATAATCAAGA3600              AGAGCAAAGCATGGATTCAAACTTAGGTATTGGAACCAGGTTTTTGTGTTTGCCCCAGTC3660              TATTTATAGAAGTGAGCTAAATGTTTATGCTTTTGGGGAGCACATTTTACAAATTTCCAA3720              GTATAGTTAAAGGAACTGCTTCTTAAACTTGAAACATGTTCCTCCTAAGGTGCTTTTCAT3780              AGAAAAAAGTCCTTCACACAGCTAGGACGTCATCTTTGACTGAATGAGCTTTAACATCCT3840              AATTACTGGTGGACTTACTTCTGGTTTCATTTTATAAAGCAAATCCCGGTGTCCCAAAGC3900              AAGGAATTTAATCATTTTGTGTGACATGAAAGTAAATCCAGTCCTGCCAATGAGAAGAAA3960              AAGACACAGCAAGTTGCAGCGTTTATAGTCTGCTTTTACATCTGAACCTCTGTTTTTGTT4020              ATTTAAGGTGAAGCAGCATCTGGGTGTGAGAGTGAAACAAGCGTCTCTGAAGACTGCTCA4080              GGGCTATCCTCTCAGAGTGACATTTTAACCACTCAGGTAAAAAGCGTGTGTGTGTGTGCA4140              CATGCGTGTGTGTGGTGTCCTTTGCATTCAGTAGTATGTATCCCACATTCTTAGGTTTGC4200              TGACATCATCTCTTTGAATTAATGGCACAATTGTTTGTGGTTCATTGTC4249                         (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 710 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      NGNGAATGTAATCCTAATATTTCNCNCCNACTTAAAAGAATACCACTCCAANGGCATCNC60                AATACATCAATCAATTGGGGAATTGGGATTTTCCCTCNCTAACATCANTGGAATAATTTC120               ATGGCATTAATTGCATGAATGTGGTTAGATTAAAAGGTGTTCATGCTAGAACTTGTAGTT180               CCATACTAGGTGATTTCAATTCCTGTGCTAAAATTAATTTGTATGATATATTNTCATTTA240               ATGGAAAGCTTCTCAAAGTATTTCATTTTCTTGGTACCATTTATCGTTTTTGAAGCAGAG300               GGATACCATGCAACATAACCTGATAAAGCTCCAGCAGGAAATGGCTGAACTAGAAGCTGT360               GTTAGAACAGCATGGGAGCCAGCCTTCTAACAGCTACCCTTCCATCATAAGTGACTCTTC420               TGCCCTTGAGGACCTGCGAAATCCAGAACAAAGCACATCAGAAAAAGGTGTGTATTGTTG480               GCCAAACACTGATATCTTAAGCAAAATTCTTTCCTTCCCCTTTATCTCCTTCTGAAGAGT540               AAGGACCTAGCTCCAACATTTTATGATCCTTGCTCAGCACATGGGTAATTATGGAGCCTT600               GGTTCTTGTCCCTGCTCACAACTAATATACCAGTCAGAGGGACCCAAGGCAGTCATTCAT660               GTTGTCATCTGAGATACCTACAACAAGTAGATGCTATGGGGAGCCCATGG710                         (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 473 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CCATTGGTGCTAGCATCTGTCTGTTGCATTGCTTGTGTTTATAAAATTCTGCCTGATATA60                CTTGTTAAAAACCAATTTGTGTATCATAGATTGATGCTTTTGAAAAAAATCAGTATTCTA120               ACCTGAATTATCACTATCAGAACAAAGCAGTAAAGTAGATTTGTTTTCTCATTCCATTTA180               AAGCAGTATTAACTTCACAGAAAAGTAGTGAATACCCTATAAGCCAGAATCCAGAAGGCC240               TTTCTGCTGACAAGTTTGAGGTGTCTGCAGATAGTTCTACCAGTAAAAATAAAGAACCAG300               GAGTGGAAAGGTAAGAAACATCAATGTAAAGATGCTGTGGTATCTGACATCTTTATTTAT360               ATTGAACTCTGATTGTTAATTTTTTTCACCATACTTTCTCCAGTTTTTTTGCATACAGGC420               ATTTATACACTTTTATTGCTCTAGGATACTTCTTTTGTTTAATCCTATATAGG473                      (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 421 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GGATAAGNTCAAGAGATATTTTGATAGGTGATGCAGTGATNAATTGNGAAAATTTNCTGC60                CTGCTTTTAATCTTCCCCCGTTCTTTCTTCCTNCCTCCCTCCCTTCCTNCCTCCCGTCCT120               TNCCTTTCCTTTCCCTCCCTTCCNCCTTCTTTCCNTCTNTCTTTCCTTTCTTTCCTGTCT180               ACCTTTCTTTCCTTCCTCCCTTCCTTTTCTTTTCTTTCTTTCCTTTCCTTTTCTTTCCTT240               TCTTTCCTTTCCTTTCTTTCTTGACAGAGTCTTGCTCTGTCACTCAGGCTGGAGTGCAGT300               GGCGTGATCTCGNCTCACTGCAACCTCTGTCTCCCAGGTTCAAGCAATTTTCCTGCCTCA360               GCCTCCCGAGTAGCTGAGATTACAGGCGCCAGCCACCACACCCAGCTACTGACCTGCTTT420               T421                                                                          (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 997 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      AAACAGCTGGGAGATATGGTGCCTCAGACCAACCCCATGTTATATGTCAACCCTGACATA60                TTGGCAGGCAACATGAATCCAGACTTCTAGGCTGTCATGCGGGCTCTTTTTTGCCAGTCA120               TTTCTGATCTCTCTGACATGAGCTGTTTCATTTATGCTTTGGCTGCCCAGCAAGTATGAT180               TTGTCCTTTCACAATTGGTGGCGATGGTTTTCTCCTTCCATTTATCTTTCTAGGTCATCC240               CCTTCTAAATGCCCATCATTAGATGATAGGTGGTACATGCACAGTTGCTCTGGGAGTCTT300               CAGAATAGAAACTACCCATCTCAAGAGGAGCTCATTAAGGTTGTTGATGTGGAGGAGCAA360               CAGCTGGAAGAGTCTGGGCCACACGATTTGACGGAAACATCTTACTTGCCAAGGCAAGAT420               CTAGGTAATATTTCATCTGCTGTATTGGAACAAACACTYTGATTTTACTCTGAATCCTAC480               ATAAAGATATTCTGGTTAACCAACTTTTAGATGTACTAGTCTATCATGGACACTTTTGTT540               ATACTTAATTAAGCCCACTTTAGAAAAATAGCTCAAGTGTTAATCAAGGTTTACTTGAAA600               ATTATTGAAACTGTTAATCCATCTATATTTTAATTAATGGTTTAACTAATGATTTTGAGG660               ATGWGGGAGTCKTGGTGTACTCTAMATGTATTATTTCAGGCCAGGCATAGTGGCTCACGC720               CTGGTAATCCCAGTAYYCMRGAGCCCGAGGCAGGTGGAGCCAGCTGAGGTCAGGAGTTCA780               AGACCTGTCTTGGCCAACATGGGNGAAACCCTGTCTTCTTCTTAAAAAANACAAAAAAAA840               TTAACTGGGTTGTGCTTAGGTGNATGCCCCGNATCCTAGTTNTTCTTGNGGGTTGAGGGA900               GGAGATCACNTTGGACCCCGGAGGGGNGGGTGGGGGNGAGCAGGNCAAAACACNGACCCA960               GCTGGGGTGGAAGGGAAGCCCACTCNAAAAAANNTTN997                                      (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 639 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      TTTTTAGGAAACAAGCTACTTTGGATTTCCACCAACACCTGTATTCATGTACCCATTTTT60                CTCTTAACCTAACTTTATTGGTCTTTTTAATTCTTAACAGAGACCAGAACTTTGTAATTC120               AACATTCATCGTTGTGTAAATTAAACTTCTCCCATTCCTTTCAGAGGGAACCCCTTACCT180               GGAATCTGGAATCAGCCTCTTCTCTGATGACCCTGAATCTGATCCTTCTGAAGACAGAGC240               CCCAGAGTCAGCTCGTGTTGGCAACATACCATCTTCAACCTCTGCATTGAAAGTTCCCCA300               ATTGAAAGTTGCAGAATCTGCCCAGAGTCCAGCTGCTGCTCATACTACTGATACTGCTGG360               GTATAATGCAATGGAAGAAAGTGTGAGCAGGGAGAAGCCAGAATTGACAGCTTCAACAGA420               AAGGGTCAACAAAAGAATGTCCATGGTGGTGTCTGGCCTGACCCCAGAAGAATTTGTGAG480               TGTATCCATATGTATCTCCCTAATGACTAAGACTTAACAACATTCTGGAAAGAGTTTTAT540               GTAGGTATTGTCAATTAATAACCTAGAGGAAGAAATCTAGAAAACAATCACAGTTCTGTG600               TAATTTAATTTCGATTACTAATTTCTGAAAATTTAGAAY639                                    (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 922 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      NCCCNNCCCCCNAATCTGAAATGGGGGTAACCCCCCCCCAACCGANACNTGGGTNGCNTA60                GAGANTTTAATGGCCCNTTCTGAGGNACANAAGCTTAAGCCAGGNGACGTGGANCNATGN120               GTTGTTTNTTGTTTGGTTACCTCCAGCCTGGGTGACAGAGCAAGACTCTGTCTAAAAAAA180               AAAAAAAAAAAAATCGACTTTAAATAGTTCCAGGACACGTGTAGAACGTGCAGGATTGCT240               ACGTAGGTAAACATATGCCATGGTGGGATAACTAGTATTCTGAGCTGTGTGCTAGAGGTA300               ACTCATGATAATGGAATATTTGATTTAATTTCAGATGCTCGTGTACAAGTTTGCCAGAAA360               ACACCACATCACTTTAACTAATCTAATTACTGAAGAGACTACTCATGTTGTTATGAAAAC420               AGGTATACCAAGAACCTTTACAGAATACCTTGCATCTGCTGCATAAAACCACATGAGGCG480               AGGCACGGTGGCGCATGCCTGTAATCGCAGCACTTTGGGAGGCCGAGGCGGGCAGATCAC540               GAGATTAGGAGATCGAGACCATCCTGGCCAGCATGGTGAAACCCCGTCTCTACTANNAAA600               TGGNAAAATTANCTGGGTGTGGTCGCGTGCNCCTGTAGTCCCAGCTACTCGTGAGGCTGA660               GGCAGGAGAATCACTTGAACCGGGGAAATGGAGGTTTCAGTGAGCAGAGATCATNCCCCT720               NCATTCCAGCCTGGCGACAGAGCAAGGCTCCGTCNCCNAAAAAATAAAAAAAAACGTGAA780               CAAATAAGAATATTTGTTGAGCATAGCATGGATGATAGTCTTCTAATAGTCAATCAATTA840               CTTTATGAAAGACAAATAATAGTTTTGCTGCTTCCTTACCTCCTTTTGTTTTGGGTTAAG900               ATTTGGAGTGTGGGCCAGGCAC922                                                     (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 867 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      GATCTATAGCTAGCCTTGGCGTCTAGAAGATGGGTGTTGAGAAGAGGGAGTGGAAAGATA60                TTTCCTCTGGTCTTAACTTCATATCAGCCTCCCCTAGACTTCCAAATATCCATACCTGCT120               GGTTATAATTAGTGGTGTTTTCAGCCTCTGATTCTGTCACCAGGGGTTTTAGAATCATAA180               ATCCAGATTGATCTTGGGAGTGTAAAAAACTGAGGCTCTTTAGCTTCTTAGGACAGCACT240               TCCTGATTTTGTTTTCAACTTCTAATCCTTTGAGTGTTTTTCATTCTGCAGATGCTGAGT300               TTGTGTGTGAACGGACACTGAAATATTTTCTAGGAATTGCGGGAGGAAAATGGGTAGTTA360               GCTATTTCTGTAAGTATAATACTATTTCTCCCCTCCTCCCTTTAACACCTCAGAATTGCA420               TTTTTACACCTAACATTTAACACCTAAGGTTTTTGCTGATGCTGAGTCTGAGTTACCAAA480               AGGTCTTTAAATTGTAATACTAAACTACTTTTATCTTTAATATCACTTTGTTCAAGATAA540               GCTGGTGATGCTGGGAAAATGGGTCTCTTTTATAACTAATAGGACCTAATCTGCTCCTAG600               CAATGTTAGCATATGAGCTAGGGATTTATTTAATAGTCGGCAGGAATCCATGTGCARCAG660               NCAAACTTATAATGTTTAAATTAAACATCAACTCTGTCTCCAGAAGGAAACTGCTGCTAC720               AAGCCTTATTAAAGGGCTGTGGCTTTAGAGGGAAGGACCTCTCCTCTGTCATTCTTCCTG780               TGCTCTTTTGTGAATCGCTGACCTCTCTATCTCCGTGAAAAGAGCACGTTCTTCTGCTGT840               ATGTAACCTGTCTTTTCTATGATCTCT867                                                (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 561 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      NAAAAACGGGGNNGGGANTGGGCCTTAAANCCAAAGGGCNAACTCCCCAACCATTNAAAA60                ANTGACNGGGGATTATTAAAANCGGCGGGAAACATTTCACNGCCCAACTAATATTGTTAA120               ATTAAAACCACCACCNCTGCNCCAAGGAGGGAAACTGCTGCTACAAGCCTTATTAAAGGG180               CTGTGGCTTTAGAGGGAAGGACCTCTCCTCTGTCATTCTTCCTGTGCTCTTTTGTGAATC240               GCTGACCTCTCTATGTCCGTGAAAAGAGCACGTTCTTCGTCTGTATGTAACCTGTCTTTT300               CTATGATCTCTTTAGGGGTGACCCAGTCTATTAAAGAAAGAAAAATGCTGAATGAGGTAA360               GTACTTGATGTTACAAACTAACCAGAGATATTCATTCAGTCATATAGTTAAAAATGTATT420               TGCTTCCTTCCATCAATGCACCACTTTCCTTAACAATGCACAAATTTTCCATGATAATGA480               GGATCATCAAGAATTATGCAGGCCTGCACTGTGGCTCATACCTATAATCCCAGCGCTTTG540               GGAGGCTGAGGCGCTTGGATC561                                                      (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 567 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      AATTTTTTGTATTTTTAGTAGAGATGAGGTTCACCATGTTGGTCTAGATCTGGTGTCGAA60                CGTCCTGACCTCAAGTGATCTGCCAGCCTCAGTCTCCCAAAGTGCTAGGATTACAGGGGT120               GAGCCACTGCGCCTGGCCTGAATGCCTAAAATATGACGTGTCTGCTCCACTTCCATTGAA180               GGAAGCTTCTCTTTCTCTTATCCTGATGGGTTGTGTTTGGTTTCTTTCAGCATGATTTTG240               AAGTCAGAGGAGATGTGGTCAATGGAAGAAACCACCAAGGTCCAAAGCGAGCAAGAGAAT300               CCCAGGACAGAAAGGTAAAGCTCCCTCCCTCAAGTTGACAAAAATCTCACCCCACCACTC360               TGTATTCCACTCCCCTTTGCAGAGATGGGCCGCTTCATTTTGTAAGACTTATTACATACA420               TACACAGTGCTAGATACTTTCACACAGGTTCTTTTTTCACTCTTCCATCCCAACCACATA480               AATAAGTATTGTCTCTACTTTATGAATGATAAAACTAAGAGATTTAGAGAGGCTGTGTAA540               TTTGGATTCCCGTCTCGGGTTCAGATC567                                                (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 633 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      TTGGCCTGATTGGTGACAAAAGTGAGATGCTCAGTCCTTGAATGACAAAGAATGCCTGTA60                GAGTTGCAGGTCCAACTACATATGCACTTCAAGAAGATCTTCTGAAATCTAGTAGTGTTC120               TGGACATTGGACTGCTTGTCCCTGGGAAGTAGCAGCAGAAATGATCGGTGGTGAACAGAA180               GAAAAAGAAAAGCTCTTCCTTTTTGAAAGTCTGTTTTTTGAATAAAAGCCAATATTCTTT240               TATAACTAGATTTTCCTTCTCTCCATTCCCCTGTCCCTCTCTCTTCCTCTCTTCTTCCAG300               ATCTTCAGGGGGCTAGAAATCTGTTGCTATGGGCCCTTCACCAACATGCCCACAGGTAAG360               AGCCTGGGAGAACCCCAGAGTTCCAGCACCAGCCTTTGTCTTACATAGTGGAGTATTATA420               AGCAAGGTCCCACGATGGGGGTTCCTCAGATTGCTGAAATGTTCTAGAGGCTATTCTATT480               TCTCTACCACTCTCCAAACAAAACAGCACCTAAATGTTATCCTATGGCAAAAAAAAACTA540               TACCTTGTCCCCCTTCTCAAGAGCATGAAGGTGGTTAATAGTTAGGATTCAGTATGTTAT600               GTGTTCAGATGGCGTTGAGCTGCTGTTAGTGCC633                                          (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 470 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      TTTGAGAGACTATCAAACCTTATACCAAGTGGCCTTATGGAGACTGATAACCAGAGTACA60                TGGCATATCAGTGGCAAATTGACTTAAAATCCATACCCCTACTATTTTAAGACCATTGTC120               CTTTGGAGCAGAGAGACAGACTCTCCCATTGAGAGGTCTTGCTATAAGCCTTCATCCGGA180               GAGTGTAGGGTAGAGGGCCTGGGTTAAGTATGCAGATTACTGCAGTGATTTTACATGTAA240               ATGTCCATTTTAGATCAACTGGAATGGATGGTACAGCTGTGTGGTGCTTCTGTGGTGAAG300               GAGCTTTCATCATTCACCCTTGGCACAGTAAGTATTGGGTGCCCTGTCAGTGTGGGAGGA360               CACAATATTCTCTCCTGTGAGCAAGACTGGCACCTGTCAGTCCCTATGGATGCCCCTACT420               GTAGCCTCAGAAGTCTTCTCTGCCCACATACCTGTGCCAAAAGACTCCAT470                         (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 517 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      GGTGGTACGTGTCTGTAGTTCCAGCTACTTGGGAGGCTGAGATGGAAGGATTGCTTGAGC60                CCAGGAGGCAGAGGTGGNANNTTACGCTGAGATCACACCACTGCACTCCAGCCTGGGTGA120               CAGAGCAAGACCCTGTCTCAAAAACAAACAAAAAAAATGATGAAGTGACAGTTCCAGTAG180               TCCTACTTTGACACTTTGAATGCTCTTTCCTTCCTGGGGATCCAGGGTGTCCACCCAATT240               GTGGTTGTGCAGCCAGATGCCTGGACAGAGGACAATGGCTTCCATGGTAAGGTGCCTCGC300               ATGTACCTGTGCTATTAGTGGGGTCCTTGTGCATGGGTTTGGTTTATCACTCATTACCTG360               GTGCTTGAGTAGCACAGTTCTTGGCACATTTTTAAATATTTGTTGAATGAATGGCTAAAA420               TGTCTTTTTGATGTTTTTATTGTTATTTGTTTTATATTGTAAAAGTAATACATGAACTGT480               TTCCATGGGGTGGGAGTAAGATATGAATGTTCATCAC517                                      (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 434 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CAGTAATCCTNAGAACTCATACGACCGGGCCCCTGGAGTCGNTGNTTNGAGCCTAGTCCN60                GGAGAATGAATTGACACTAATCTCTGCTTGTGTTCTCTGTCTCCAGCAATTGGGCAGATG120               TGTGAGGCACCTGTGGTGACCCGAGAGTGGGTGTTGGACAGTGTAGCACTCTACCAGTGC180               CAGGAGCTGGACACCTACCTGATACCCCAGATCCCCCACAGCCACTACTGACTGCAGCCA240               GCCACAGGTACAGAGCCACAGGACCCCAAGAATGAGCTTACAAAGTGGCCTTTCCAGGCC300               CTGGGAGCTCCTCTCACTCTTCAGTCCTTCTACTGTCCTGGCTACTAAATATTTTATGTA360               CATCAGCCTGAAAAGGACTTCTGGCTATGCAAGGGTCCCTTAAAGATTTTCTGCTTGAAG420               TCTCCCTTGGAAAT434                                                             (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      GATAAATTAAAACTGCGACTGCGCGGCGTG30                                              (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      GTAGTAGAGTCCCGGGAAAGGGACAGGGGG30                                              (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      ATATATATATGTTTTTCTAATGTGTTAAAG30                                              (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      GTAAGTCAGCACAAGAGTGTATTAATTTGG30                                              (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      TTTCTTTTTCTCCCCCCCCTACCCTGCTAG30                                              (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      GTAAGTTTGAATGTGTTATGTGGCTCCATT30                                              (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      AGCTACTTTTTTTTTTTTTTTTTGAGACAG30                                              (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      GTAAGTGCACACCACCATATCCAGCTAAAT30                                              (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      AATTGTTCTTTCTTTCTTTATAATTTATAG30                                              (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      GTATATAATTTGGTAATGATGCTAGGTTGG30                                              (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      GAGTGTGTTTCTCAAACAATTTAATTTCAG30                                              (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      GTAAGTGTTGAATATCCCAAGAATGACACT30                                              (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      AAACATAATGTTTTCCCTTGTATTTTACAG30                                              (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      GTAAAACCATTTGTTTTCTTCTTCTTCTTC30                                              (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      TGCTTGACTGTTCTTTACCATACTGTTTAG30                                              (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      GTAAGGGTCTCAGGTTTTTTAAGTATTTAA30                                              (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      TGATTTATTTTTTGGGGGGAAATTTTTTAG30                                              (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      GTGAGTCAAAGAGAACCTTTGTCTATGAAG30                                              (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      TCTTATTAGGACTCTGTCTTTTCCCTATAG30                                              (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      GTAATGGCAAAGTTTGCCAACTTAACAGGC30                                              (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      GAGTACCTTGTTATTTTTGTATATTTTCAG30                                              (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      GTATTGGAACCAGGTTTTTGTGTTTGCCCC30                                              (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      ACATCTGAACCTCTGTTTTTGTTATTTAAG30                                              (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      AGGTAAAAAGCGTGTGTGTGTGTGCACATG30                                              (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      CATTTTCTTGGTACCATTTATCGTTTTTGA30                                              (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      GTGTGTATTGTTGGCCAAACACTGATATCT30                                              (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      AGTAGATTTGTTTTCTCATTCCATTTAAAG30                                              (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      GTAAGAAACATCAATGTAAAGATGCTGTGG30                                              (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      ATGGTTTTCTCCTTCCATTTATCTTTCTAG30                                              (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      GTAATATTTCATCTGCTGTATTGGAACAAA30                                              (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      TGTAAATTAAACTTCTCCCATTCCTTTCAG30                                              (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      GTGAGTGTATCCATATGTATCTCCCTAATG30                                              (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      ATGATAATGGAATATTTGATTTAATTTCAG30                                              (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      GTATACCAAGAACCTTTACAGAATACCTTG30                                              (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      CTAATCCTTTGAGTGTTTTTCATTCTGCAG30                                              (2) INFORMATION FOR SEQ ID NO:70:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                      GTAAGTATAATACTATTTCTCCCCTCCTCC30                                              (2) INFORMATION FOR SEQ ID NO:71:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                      TGTAACCTGTCTTTTCTATGATCTCTTTAG30                                              (2) INFORMATION FOR SEQ ID NO:72:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                      GTAAGTACTTGATGTTACAAACTAACCAGA30                                              (2) INFORMATION FOR SEQ ID NO:73:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                      TCCTGATGGGTTGTGTTTGGTTTCTTTCAG30                                              (2) INFORMATION FOR SEQ ID NO:74:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                      GTAAAGCTCCCTCCCTCAAGTTGACAAAAA30                                              (2) INFORMATION FOR SEQ ID NO:75:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                      CTGTCCCTCTCTCTTCCTCTCTTCTTCCAG30                                              (2) INFORMATION FOR SEQ ID NO:76:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                      GTAAGAGCCTGGGAGAACCCCAGAGTTCCA30                                              (2) INFORMATION FOR SEQ ID NO:77:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                      AGTGATTTTACATGTAAATGTCCATTTTAG30                                              (2) INFORMATION FOR SEQ ID NO:78:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                      GTAAGTATTGGGTGCCCTGTCAGTGTGGGA30                                              (2) INFORMATION FOR SEQ ID NO:79:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                      TTGAATGCTCTTTCCTTCCTGGGGATCCAG30                                              (2) INFORMATION FOR SEQ ID NO:80:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                      GTAAGGTGCCTCGCATGTACCTGTGCTATT30                                              (2) INFORMATION FOR SEQ ID NO:81:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                      CTAATCTCTGCTTGTGTTCTCTGTCTCCAG30                                              (2) INFORMATION FOR SEQ ID NO:82:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                      CysProIleCysLeuGluLeuIleLysGluProValSerThrLysCys                              151015                                                                        AspHisIlePheCysLysPheCysMetLeuLysLeuLeuAsnGlnLys                              202530                                                                        LysGlyProSerGlnCysProLeuCysLys                                                3540                                                                          (2) INFORMATION FOR SEQ ID NO:83:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                      CysProIleCysLeuGluLeuLeuLysGluProValSerAlaAspCys                              151015                                                                        AsnHisSerPheCysArgAlaCysIleThrLeuAsnTyrGluSerAsn                              202530                                                                        ArgAsnThrAspGlyLysGlyAsnCysProValCysArg                                       354045                                                                        (2) INFORMATION FOR SEQ ID NO:84:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                      CysProIleCysLeuAspMetLeuLysAsnThrMetThrThrLysGlu                              151015                                                                        CysLeuHisArgPheCysSerAspCysIleValThrAlaLeuArgSer                              202530                                                                        GlyAsnLysGluCysProThrCysArg                                                   3540                                                                          (2) INFORMATION FOR SEQ ID NO:85:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                      CysProValCysLeuGlnTyrPheAlaGluProMetMetLeuAspCys                              151015                                                                        GlyHisAsnIleCysCysAlaCysLeuAlaArgCysTrpGlyThrAla                              202530                                                                        CysThrAsnValSerCysProGlnCysArg                                                3540                                                                          __________________________________________________________________________

What is claimed is:
 1. An isolated DNA coding for a BRCA1 polypeptide,said polypeptide having the amino acid sequence set forth in SEQ IDNO:2.
 2. The isolated DNA of claim 1, wherein said DNA has thenucleotide sequence set forth in SEQ ID NO:1.
 3. The isolated DNA ofclaim 1 which contains BRCA1 regulatory sequences.
 4. The isolated DNAof claim 2 which contains BRCA1 regulatory sequences.
 5. An isolated DNAhaving at least 15 nucleotides of the DNA of claim
 1. 6. An isolated DNAhaving at least 15 nucleotides of the DNA of claim
 2. 7. An isolated DNAselected from the group consisting of:(a) a DNA having the nucleotidesequence set forth in SEQ ID NO:1 having T at nucleotide position 4056;(b) a DNA having the nucleotide sequence set forth in SEQ ID NO:1 havingan extra C at nucleotide position 5385; (c) a DNA having the nucleotidesequence set forth in SEQ ID NO: 1 having G at nucleotide position 5443;and, (d) a DNA having the nucleotide sequence set forth in SEQ ID NO:1having 11 base pairs at nucleotide positions 189-199 deleted.
 8. Areplicative cloning vector which comprises the isolated DNA of claim 1or parts thereof and a replicon operative in a host cell.
 9. Areplicative cloning vector which comprises the isolated DNA of claim 2or parts thereof and a replicon operative in a host cell.
 10. Anexpression system which comprises the isolated DNA of claim 1 or partsthereof operably linked to suitable control sequences.
 11. An expressionsystem which comprises the isolated DNA of claim 2 or parts thereofoperably linked to suitable control sequences.
 12. Host cellstransformed with the expression system of claim
 10. 13. Host cellstransformed with the expression system of claim
 11. 14. A method ofproducing BRCA1 polypeptide which comprises culturing the cells of claim12 under conditions effective for the production of saod BRCA1polypeptide and harvesting the BRCA1 polypeptide.
 15. A method ofproducing BRCA1 polypeptide which comprises culturing the cells of claim13 under conditions effective for the producton of said BRCA1polypeptide and harvesting the BRCA1 polypeptide.
 16. A pair ofsingle-stranded DNA primers for determination of a nuycleotide sequenceof a BRCA1 gene by a polymerase chin reaction, the sequence of saidprimers being derived from human chromosomne 17q, wherein the use ofsaid primers in a polymerase chain reaction results in the synthesis ofDNA having all or part of the sequence of the BRCA1 gene.
 17. The pairof primers of claim 16 wherin said BRCA1 gene has the nucleotidesequence set forth in SEQ ID NO:1.
 18. A kit for detecting mutations inthe BRCA1 gene resulting in a susceptibility to breast and ovarianncancers comprising at least one oligonucleotide prime specific for aBRCA1 gene mutation and instructions relatiing to detectiong mutationsin the BRCA1 gene.
 19. A kit for detecting mutations in the BRCA1 generesulting in a sisceptibility to breast and ovarian cancers comprisingat least one allele-specific oligimucleotide probe for a BRCA1 genemutation and instructions relating to detecting mutations in the BRCA1gene.
 20. A method for screening potential cancer therapeutics whichcomprises: growing a transformed eukaryotic host cell containing analtered BRCA1 gene causing cancer in the presence of a compoundsuspected of being a cancer therapeutic, growing said transformedeukaryotic host cell in the absence of said compound, determining therate of growth of said host cell in the presence of said compound andthe rate of growth of said host cell in the absence of said compound andcomparing the growth rate of said host cells, wherein a slower rate ofgrowth of said host cell in the presence of said compound is indicativeof a cancer therapeutic.